scholarly journals Location bias contributes to functionally selective responses of biased CXCR3 agonists

2022 ◽  
Author(s):  
Dylan Scott Eiger ◽  
Noelia Boldizsar ◽  
Christopher Cole Honeycutt ◽  
Julia Gardner ◽  
Stephen Kirchner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cd8 T Cells ◽  
Chemokine Receptors ◽  
Receptor Internalization ◽  
Contact Hypersensitivity ◽  
Transcriptional Responses ◽  
Endosomal Signaling ◽  
Specific Manner ◽  
Biased Signaling ◽  
G Protein Coupled

Some G protein-coupled receptor (GPCR) ligands act as biased agonists which preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the chemokine GPCR CXCR3. The signaling profile of CXCR3 changed as it trafficked from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling was critical for biased activation of G proteins, β-arrestins, and ERK1/2. In CD8+ T cells, the chemokines promoted unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation was dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

scholarly journals Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR

Science ◽  
2020 ◽  
Vol 367 (6480) ◽  
pp. 888-892 ◽  
Author(s):  
Laura M. Wingler ◽  
Meredith A. Skiba ◽  
Conor McMahon ◽  
Dean P. Staus ◽  
Alissa L. W. Kleinhenz ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Chemokine Receptors ◽  
Binding Pocket ◽  
Protein Signaling ◽  
Downstream Signaling ◽  
The Family ◽  
Biased Signaling ◽  
Sodium Binding ◽  
G Protein Coupled

Biased agonists of G protein–coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly β-arrestin–biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating β-arrestin but not heterotrimeric Gq protein signaling.

scholarly journals Biased Signaling: Distinct Ligand-directed Plasma Membrane Signalosomes Using a Common RGS/G protein Core

2019 ◽  
Author(s):  
Timothy J. Ross-Elliott ◽  
Justin Watkins ◽  
Xiaoyi Shan ◽  
Fei Lou ◽  
Bernd Dreyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Protein Complex ◽  
Heterotrimeric G Protein ◽  
Unknown Mechanism ◽  
Protein Core ◽  
Protein System ◽  
Biased Signaling ◽  
G Protein Coupled

Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: sterol-dependent domains, possibly lipid rafts, and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

scholarly journals Distinct Mechanisms of Agonist-induced Endocytosis for Human Chemokine Receptors CCR5 and CXCR4

2003 ◽  
Vol 14 (8) ◽  
pp. 3305-3324 ◽  
Author(s):  
Sundararajan Venkatesan ◽  
Jeremy J. Rose ◽  
Robert Lodge ◽  
Philip M. Murphy ◽  
John F. Foley
Keyword(s):  
Plasma Membrane ◽  
Chemokine Receptors ◽  
G Protein Coupled Receptors ◽  
Membrane Rafts ◽  
Expression Systems ◽  
Receptor Endocytosis ◽  
Plasma Membrane Rafts ◽  
G Protein Coupled ◽  
Transmission Of Disease ◽  
Dependent Pathway

Desensitization of the chemokine receptors, a large class of G protein–coupled receptors, is mediated in part by agonist-driven receptor endocytosis. However, the exact pathways have not been fully defined. Here we demonstrate that the rate of ligand-induced endocytosis of CCR5 in leukocytes and expression systems is significantly slower than that of CXCR4 and requires prolonged agonist treatment, suggesting that these two receptors use distinct mechanisms. We show that the C-terminal domain of CCR5 is the determinant of its slow endocytosis phenotype. When the C-tail of CXCR4 was exchanged for that of CCR5, the resulting CXCR4-CCR5 (X4-R5) chimera displayed a CCR5-like trafficking phenotype. We found that the palmitoylated cysteine residues in this domain anchor CCR5 to plasma membrane rafts. CXCR4 and a C-terminally truncated CCR5 mutant (CCR5-KRFX) lacking these cysteines are not raft associated and are endocytosed by a clathrin-dependent pathway. Genetic inhibition of clathrin-mediated endocytosis demonstrated that a significant fraction of ligand-occupied CCR5 trafficked by clathrin-independent routes into caveolin-containing vesicular structures. Thus, the palmitoylated C-tail of CCR5 is the major determinant of its raft association and endocytic itineraries, differentiating it from CXCR4 and other chemokine receptors. This novel feature of CCR5 may modulate its signaling potential and could explain its preferential use by HIV for person-to-person transmission of disease.

scholarly journals Subcellular Trafficking of the TRH Receptor: Effect of Phosphorylation

2009 ◽  
Vol 23 (9) ◽  
pp. 1466-1478 ◽  
Author(s):  
Brian W. Jones ◽  
Patricia M. Hinkle
Keyword(s):  
Plasma Membrane ◽  
Receptor Trafficking ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Epitope Tag ◽  
Surface Receptors ◽  
Surface Receptor ◽  
G Protein Coupled ◽  
Trh Receptor

Abstract Activation of the G protein-coupled TRH receptor leads to its phosphorylation and internalization. These studies addressed the fundamental question of whether phosphorylation regulates receptor trafficking or endosomal localization regulates the phosphorylation state of the receptor. Trafficking of phosphorylated and dephosphorylated TRH receptors was characterized using phosphosite-specific antibody after labeling surface receptors with antibody to an extracellular epitope tag. Rab5 and phosphoreceptor did not colocalize at the plasma membrane immediately after TRH addition but overlapped extensively by 15 min. Dominant-negative Rab5-S34N inhibited receptor internalization. Later, phosphoreceptor was in endosomes containing Rab5 and Rab4. Dephosphorylated receptor colocalized with Rab4 but not with Rab5. Dominant-negative Rab4, -5, or -11 did not affect receptor phosphorylation or dephosphorylation, showing that phosphorylation determines localization in Rab4+/Rab5− vesicles and not vice versa. No receptor colocalized with Rab7; a small amount of phosphoreceptor colocalized with Rab11. To characterize recycling, surface receptors were tagged with antibody, or surface receptors containing an N-terminal biotin ligase acceptor sequence were labeled with biotin. Most recycling receptors did not return to the plasma membrane for more than 2 h after TRH was removed, whereas the total cell surface receptor density was largely restored in less than 1 h, indicating that recruited receptors contribute heavily to early repopulation of the plasma membrane.

scholarly journals GRKs as Modulators of Neurotransmitter Receptors

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 52
Author(s):  
Eugenia V. Gurevich ◽  
Vsevolod V. Gurevich
Keyword(s):  
G Protein ◽  
General Model ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Neurotransmitter Receptors ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Receptor Kinase ◽  
G Protein Coupled ◽  
Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

Abstract 626: Interaction Between The Ang-(1-7) Receptor Mas And The Bradykinin B2 Receptor: Functional Implications

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Bruno Cerrato ◽  
Oscar Carretero ◽  
Hernán Grecco ◽  
Mariela M Gironacci
Keyword(s):  
Plasma Membrane ◽  
Binding Assays ◽  
Transfected Cells ◽  
Oligomer Formation ◽  
Early Endosomes ◽  
Functional Implications ◽  
Functional Consequences ◽  
Ligand Stimulation ◽  
G Protein Coupled ◽  
Fluorescence Energy

G protein-coupled receptors (R) exist as homo- or hetero-oligomers, which is essential for receptor function. Since BK actions were blocked by a Mas R antagonist or that Ang-(1-7) responses disappeared when the BK receptor B2 was blocked, we hypothesized that Mas and B2 Rs on the plasma membrane may interact through hetero-oligomer formation. Our aim was to investigate the existence of heteromerization between Mas and B2 Rs by the fluorescence energy transfer (FRET) technique and the functional consequences of this oligomer formation. HEK293T cells were transfected with the coding sequence for Mas R fused to YFP and B2 R fused to CFP. After 48 h cells were incubated in the absence and presence of 1 μM Ang-(1-7) or BK during 15 min and interaction between Mas and B2 R was evaluated by FRET. Functional consequences of this interaction were determined by ligand binding assays. A positive FRET was observed in cells cotransfected with MasR-YFP and B2R-CFP, suggesting that both Mas and B2 Rs interact by a hetero-oligomer formation in a constitutive manner. This hetero-oligomer was not altered by the agonist because FRET was not modified when the cells were stimulated with BK or Ang-(1-7). Ang-(1-7) or BK induced internalization of this hetero-oligomer into early endosomes since MasR-YFP or B2R-CFP colocalized with Rab-5, an early endosome marker, after ligand stimulation. When MasR-YFP plus B2R-CFP transfected cells were stimulated with Ang-(1-7) there was a decrease of 82±6% in Mas R and 58±4% in B2 R present in the plasma membrane. Conversely, when MasR-YFP plus B2R-CFP transfected cells were stimulated with BK there was a decrease of 91±4% in B2 R and 53±3% in Mas R in the plasma membrane. This result clearly demonstrates that in co-expressing cells of both receptors the selective stimulation of one of the GPCRs promotes co-internalization of both receptors. We conclude that Mas and B2 Rs constitutively interact through an hetero-oligomer formation at the plasma membrane which may explain the cross-talk between Ang-(1-7) and BK. This hetero-oligomer is internalized upon stimulation with either Ang-(1-7) or BK, leading to a decrease in the number of Rs present in the membrane.

Protein kinase C(alpha) actively downregulates through caveolae-dependent traffic to an endosomal compartment

2000 ◽  
Vol 113 (14) ◽  
pp. 2575-2584
Author(s):  
C. Prevostel ◽  
V. Alice ◽  
D. Joubert ◽  
P.J. Parker
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Initial Step ◽  
Receptor Internalization ◽  
Similar Process ◽  
Receptor Desensitization ◽  
Kinase C ◽  
Annexin I ◽  
Pkc Alpha

Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC(alpha) from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC(α) protein. It is established that PKC(α) is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC(alpha) downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC(α) delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC(alpha) is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC(alpha) traffic and down regulation. Consistent with this, the endosomes where PKC(alpha) is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC(alpha) involves active delivery to endosomes via a caveolae mediated process.

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