scholarly journals NHERF2 Specifically Interacts with LPA2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation

2004 ◽  
Vol 24 (11) ◽  
pp. 5069-5079 ◽  
Author(s):  
Yong-Seok Oh ◽  
Nam Won Jo ◽  
Jung Woong Choi ◽  
Hyeon Soo Kim ◽  
Sang-Won Seo ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Phospholipase C ◽  
Pdz Domain ◽  
The Other ◽  
Binding Motif ◽  
Lpa Receptor ◽  
Erk Activation ◽  
Downstream Signaling ◽  
Receptor Isoforms ◽  
G Protein Coupled

ABSTRACT Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA2, but not the other LPA receptor isoforms, specifically interacts with Na+/H+ exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA2 and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-β (PLC-β) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA2 or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA2 to PLC-β3 to form a complex, and the other PLC-β isozymes were not included in the protein complex. Consistently, LPA2-mediated PLC-β activation was specifically inhibited by the gene silencing of PLC-β3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA2, NHERF2, and PLC-β3 may play a key role in the LPA2-mediated PLC-β signaling pathway.

scholarly journals The Effects of Apelin and Elabela Ligands on Apelin Receptor Distinct Signaling Profiles

2021 ◽  
Vol 12 ◽  
Author(s):  
Yunlu Jiang ◽  
Maocai Yan ◽  
Chunmei Wang ◽  
Qinqin Wang ◽  
Xiaoyu Chen ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Signaling Pathways ◽  
G Protein ◽  
Peptide Ligands ◽  
Erk Activation ◽  
Downstream Signaling ◽  
Apelin Receptor ◽  
Clathrin Adaptor ◽  
And Function ◽  
G Protein Coupled

Apelin and Elabela are endogenous peptide ligands for Apelin receptor (APJ), a widely expressed G protein-coupled receptor. They constitute a spatiotemporal dual ligand system to control APJ signal transduction and function. We investigated the effects of Apelin-13, pGlu1-apelin-13, Apelin-17, Apelin-36, Elabela-21 and Elabela-32 peptides on APJ signal transduction. Whether different ligands are biased to different APJ mediated signal transduction pathways was studied. We observed the different changes of G protein dependent and β-arrestin dependent signaling pathways after APJ was activated by six peptide ligands. We demonstrated that stimulation with APJ ligands resulted in dose-dependent increases in both G protein dependent [cyclic AMP (cAMP), Ca2+ mobilization, and the early phase extracellular related kinase (ERK) activation] and β-arrestin dependent [GRKs, β-arrestin 1, β-arrestin 2, and β2 subunit of the clathrin adaptor AP2] signaling pathways. However, the ligands exhibited distinct signaling profiles. Elabela-32 showed a >1000-fold bias to the β-statin-dependent signaling pathway. These data provide that Apelin-17 was biased toward β-arrestin dependent signaling. Eabela-21 and pGlu1-Apelin-13 exhibited very distinct activities on the G protein dependent pathway. The activity profiles of these ligands could be valuable for the development of drugs with high selectivity for specific APJ downstream signaling pathways.

scholarly journals Cartilage oligomeric matrix protein is an endogenous β-arrestin-2-selective allosteric modulator of AT1 receptor counteracting vascular injury

Cell Research ◽  
2021 ◽  
Author(s):  
Yi Fu ◽  
Yaqian Huang ◽  
Zhao Yang ◽  
Yufei Chen ◽  
Jingang Zheng ◽  
...  
Keyword(s):  
Cartilage Oligomeric Matrix Protein ◽  
Matrix Protein ◽  
Therapeutic Strategies ◽  
At1 Receptor ◽  
Extracellular Matrix Protein ◽  
Binding Motif ◽  
Abdominal Aortic ◽  
Novel Therapeutic Strategies ◽  
G Protein Coupled

AbstractCompelling evidence has revealed that biased activation of G protein-coupled receptor (GPCR) signaling, including angiotensin II (AngII) receptor type 1 (AT1) signaling, plays pivotal roles in vascular homeostasis and injury, but whether a clinically relevant endogenous biased antagonism of AT1 signaling exists under physiological and pathophysiological conditions has not been clearly elucidated. Here, we show that an extracellular matrix protein, cartilage oligomeric matrix protein (COMP), acts as an endogenous allosteric biased modulator of the AT1 receptor and its deficiency is clinically associated with abdominal aortic aneurysm (AAA) development. COMP directly interacts with the extracellular N-terminus of the AT1 via its EGF domain and inhibits AT1-β-arrestin-2 signaling, but not Gq or Gi signaling, in a selective manner through allosteric regulation of AT1 intracellular conformational states. COMP deficiency results in activation of AT1a-β-arrestin-2 signaling and subsequent exclusive AAA formation in response to AngII infusion. AAAs in COMP–/– or ApoE–/– mice are rescued by AT1a or β-arrestin-2 deficiency, or the application of a peptidomimetic mimicking the AT1-binding motif of COMP. Explorations of the endogenous biased antagonism of AT1 receptor or other GPCRs may reveal novel therapeutic strategies for cardiovascular diseases.

scholarly journals MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

2020 ◽  
Author(s):  
Ryan Ard ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Michael Phan ◽  
Radoslav Zinoviev ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Pdz Domain ◽  
Diacylglycerol Kinase ◽  
Binding Motif ◽  
Membrane Blebbing ◽  
Rhoa Activity ◽  
C Terminus ◽  
Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

scholarly journals Constitutive signal bias mediated by the human GHRHR splice variant 1

2021 ◽  
Vol 118 (40) ◽  
pp. e2106606118
Author(s):  
Zhaotong Cong ◽  
Fulai Zhou ◽  
Chao Zhang ◽  
Xinyu Zou ◽  
Huibing Zhang ◽  
...  
Keyword(s):  
Splice Variant ◽  
Human Growth ◽  
Bound State ◽  
G Protein Coupled Receptors ◽  
Cancer Cell Proliferation ◽  
Downstream Signaling ◽  
Functional Studies ◽  
Dynamics Simulations ◽  
Protein Molecular Dynamics ◽  
G Protein Coupled

Alternative splicing of G protein–coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone–releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

scholarly journals Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

2005 ◽  
Vol 168 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Doris Meder ◽  
Anna Shevchenko ◽  
Kai Simons ◽  
Joachim Füllekrug
Keyword(s):  
Free Surface ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Binding Motif ◽  
Membrane Domains ◽  
Regulatory Factor ◽  
Pdz Protein

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine

1993 ◽  
Vol 265 (5) ◽  
pp. F605-F623 ◽  
Author(s):  
R. Garza-Quintero ◽  
J. M. Weinberg ◽  
J. Ortega-Lopez ◽  
J. A. Davis ◽  
M. A. Venkatachalam
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Cell Structure ◽  
Proximal Tubules ◽  
Atp Depletion ◽  
The Other ◽  
Antimycin A ◽  
Phospholipid Hydrolysis ◽  
Hydrolysis Of

Increases of intracellular free Ca2+ (Caf) may mediate phospholipid hydrolysis and disintegration in energy-compromised cells; on the other hand, glycine and related amino acids preserve structure. We have examined the effects of increased Caf on phospholipids and structure in ATP-depleted cells, as well as how these actions may be modified by glycine. Incubation of isolated proximal tubules with antimycin A led to ATP depletion, delayed increases of Caf to micromolar levels, polyphosphoinositide (PPI) hydrolysis by phospholipase C, and generalized disintegration of cell structure. Glycine inhibited PPI hydrolysis and preserved cell structure in entirety but did not apparently modify the Caf increases. When overwhelming increases of Caf were induced by the additional presence of a Ca2+ ionophore, glycine did not inhibit either the hydrolysis of PPI or disruption of mitochondria and microvilli. However, the cells remained integrated and unbroken. Incubation in low-Ca2+ medium prevented Caf increases, inhibited PPI hydrolysis, and preserved the structure of mitochondria and microvilli. Nevertheless, there was lethal damage by disintegration of all other membranes. This damage was prevented specifically and completely by glycine. Thus compartments of cells were shown to be differentially susceptible to injury from increased Caf or lack of glycine. Although damage by either factor occurs by distinct mechanisms, glycine also appears to have effects that suppress the deleterious effects of Ca2+ so long as Caf increases are not overwhelming. Our results also suggest that the PPI have a major structural role, which may be compromised by Caf increase during ATP depletion.

scholarly journals Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader

2020 ◽  
Vol 61 (8) ◽  
pp. 1244-1251 ◽  
Author(s):  
Manisha Ray ◽  
Kazufumi Nagai ◽  
Yasuyuki Kihara ◽  
Amanda Kussrow ◽  
Michael N. Kammer ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Lysophosphatidic Acid ◽  
Specific Binding ◽  
Test Case ◽  
Free Solution ◽  
Label Free ◽  
Adhesive Properties ◽  
Lpa Receptor ◽  
System A ◽  
G Protein Coupled

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

scholarly journals Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins

2000 ◽  
Vol 74 (20) ◽  
pp. 9680-9693 ◽  
Author(s):  
Siu Sylvia Lee ◽  
Britt Glaunsinger ◽  
Fiamma Mantovani ◽  
Lawrence Banks ◽  
Ronald T. Javier
Keyword(s):  
High Risk ◽  
Cellular Proliferation ◽  
Pdz Domain ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Human Papillomaviruses ◽  
Binding Motif ◽  
Cellular Factor ◽  
Domain Protein ◽  
Hpv 18

ABSTRACT A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

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