Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

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A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m113.497826 ◽

2013 ◽

Vol 288 (38) ◽

pp. 27327-27342 ◽

Cited By ~ 15

Author(s):

Wayne Croft ◽

Claire Hill ◽

Eilish McCann ◽

Michael Bond ◽

Manuel Esparza-Franco ◽

...

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Gpcr Signaling ◽

Additional Layer ◽

Physiological Relevance ◽

Gtpase Cycle ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

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Adhesion G Protein–Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms

Molecular Pharmacology ◽

10.1124/mol.115.098749 ◽

2015 ◽

Vol 88 (3) ◽

pp. 617-623 ◽

Cited By ~ 30

Author(s):

Kelly R. Monk ◽

Jörg Hamann ◽

Tobias Langenhan ◽

Saskia Nijmeijer ◽

Torsten Schöneberg ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

In Vitro Pharmacology ◽

G Protein Coupled

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Imaging of persistent cAMP signaling by internalized G protein-coupled receptors

Journal of Molecular Endocrinology ◽

10.1677/jme-10-0014 ◽

2010 ◽

Vol 45 (1) ◽

pp. 1-8 ◽

Cited By ~ 47

Author(s):

Davide Calebiro ◽

Viacheslav O Nikolaev ◽

Martin J Lohse

Keyword(s):

G Protein ◽

Intracellular Signaling ◽

Current Model ◽

Living Cells ◽

G Protein Coupled Receptors ◽

Membrane Receptors ◽

Optical Methods ◽

Camp Signaling ◽

Gpcr Signaling ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR–cAMP signaling pathway to accommodate receptor signaling at endosomes.

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Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

Journal of Cell Science ◽

10.1242/jcs.113.13.2463 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2463-2470 ◽

Cited By ~ 1

Author(s):

F. Santini ◽

R.B. Penn ◽

A.W. Gagnon ◽

J.L. Benovic ◽

J.H. Keen

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Coated Pits ◽

Over Expression ◽

Physiological Conditions ◽

A Cell ◽

G Protein Coupled ◽

Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

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G-protein-coupled-receptor activation of the smooth muscle calponin gene

Biochemical Journal ◽

10.1042/bj3570587 ◽

2001 ◽

Vol 357 (2) ◽

pp. 587-592 ◽

Cited By ~ 9

Author(s):

Nickolai O. DULIN ◽

Sergei N. ORLOV ◽

Chad M. KITCHEN ◽

Tatyana A. VOYNO-YASENETSKAYA ◽

Joseph M. MIANO

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

G Protein ◽

Transcriptional Activation ◽

Fold Increase ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Mitogen Activated Protein Kinase ◽

G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

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Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Current Opinion in Chemical Biology ◽

10.1016/j.cbpa.2011.03.010 ◽

2011 ◽

Vol 15 (3) ◽

pp. 392-398 ◽

Cited By ~ 34

Author(s):

Kelly A Daggett ◽

Thomas P Sakmar

Keyword(s):

G Protein ◽

Molecular Probes ◽

G Protein Coupled Receptors ◽

Site Specific ◽

G Protein Coupled

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Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20156101019 ◽

2015 ◽

Vol 61 (1) ◽

pp. 19-29 ◽

Cited By ~ 1

Author(s):

A.O. Shpakov ◽

E.A. Shpakova

Keyword(s):

G Protein ◽

Intracellular Signaling ◽

High Efficiency ◽

Clinical Medicine ◽

Inflammatory Diseases ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Coupled

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

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Melatonin promotes sleep by activating the BK channel in C. elegans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010928117 ◽

2020 ◽

Vol 117 (40) ◽

pp. 25128-25137

Author(s):

Longgang Niu ◽

Yan Li ◽

Pengyu Zong ◽

Ping Liu ◽

Yuan Shui ◽

...

Keyword(s):

G Protein ◽

Neurotransmitter Release ◽

Expression System ◽

Molecular Target ◽

Bk Channel ◽

G Protein Coupled Receptors ◽

Wild Type ◽

Heterologous Expression System ◽

C Elegans ◽

G Protein Coupled

Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2. A peptide acting to release free Gβγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.

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