Cellular and subcellular localization of G-protein receptor kinases, arrestins and G-proteins: Implications for receptor regulation

The age-related decline in β-adrenergic receptor (β-AR)-mediated vasorelaxation is associated with desensitization of β-ARs without significant downregulation. The primary mode of this homologous β-AR desensitization, in general, is via G protein receptor kinases (GRK). Therefore, we hypothesize that age-related changes in GRKs are causative to this etiology in rat aorta. Herein, we investigate the activity and cellular distribution (cytoplasmic vs. membrane) of several GRK isoforms and β-arrestin proteins. GRK activity was assessed in extracts from aortic tissue of 6-wk, 6-mo, 12-mo, and 24-mo-old male Fischer-344 rats using a rhodopsin phosphorylation assay. We also performed immunoblots on lysates from aorta with specific antibodies to GRK-2, -3, -5, and β-arrestin-1. Results show an age-related increase in GRK activity. Furthermore, expression of GRK-2 (cytoplasmic and membrane), GRK-3 (cytoplasmic and membrane), and β-arrestin (soluble) increased with advancing age, whereas GRK-5 (membrane) expression remained unchanged. These results suggest that age is associated with increased activity and expression of specific GRKs. This increase likely results in enhanced phosphorylation and desensitization of β-ARs. These biochemical changes are consistent with observed aging physiology.

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Nuclear GPCRs in cardiomyocytes: an insider's view of β-adrenergic receptor signaling

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00657.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. H1754-H1764 ◽

Cited By ~ 39

Author(s):

George Vaniotis ◽

Bruce G. Allen ◽

Terence E. Hébert

Keyword(s):

Subcellular Localization ◽

G Protein ◽

G Proteins ◽

Adrenergic Receptor ◽

Physiological State ◽

Signaling Cascades ◽

Special Focus ◽

The Novel ◽

Receptor Signaling ◽

G Protein Coupled

In recent years, we have come to appreciate the complexity of G protein-coupled receptor signaling in general and β-adrenergic receptor (β-AR) signaling in particular. Starting originally from three β-AR subtypes expressed in cardiomyocytes with relatively simple, linear signaling cascades, it is now clear that there are large receptor-based networks which provide a rich and diverse set of responses depending on their complement of signaling partners and the physiological state. More recently, it has become clear that subcellular localization of these signaling complexes also enriches the diversity of phenotypic outcomes. Here, we review our understanding of the signaling repertoire controlled by nuclear β-AR subtypes as well our understanding of the novel roles for G proteins themselves in the nucleus, with a special focus, where possible, on their effects in cardiomyocytes. Finally, we discuss the potential pathological implications of alterations in nuclear β-AR signaling.

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Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors

Biochemical Society Transactions ◽

10.1042/bst0280419 ◽

2000 ◽

Vol 28 (4) ◽

pp. 419-426 ◽

Cited By ~ 18

Author(s):

K. DeFea ◽

F. Schmidlin ◽

O. Déry ◽

E. F. Grady ◽

N. W. Bunnett

Keyword(s):

Cell Surface ◽

G Protein ◽

G Proteins ◽

Extracellular Fluid ◽

Neutral Endopeptidase ◽

Neuropeptide Receptors ◽

Biological Responses ◽

Protein Receptor ◽

Proteinase Activated Receptors ◽

Neurokinin 1

Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with β-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a β-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.

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G-protein receptor kinases 2, 5 and 6 redundantly modulate Smoothened-GATA transcriptional crosstalk in fetal mouse hearts

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2018.06.009 ◽

2018 ◽

Vol 121 ◽

pp. 60-68 ◽

Cited By ~ 3

Author(s):

Antonietta Franco ◽

Lihong Zhang ◽

Scot J. Matkovich ◽

Attila Kovacs ◽

Gerald W. Dorn

Keyword(s):

G Protein ◽

Fetal Mouse ◽

Protein Receptor ◽

Receptor Kinases

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Agonist-induced formation of unproductive receptor-G12complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2003787117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21723-21730

Author(s):

Najeah Okashah ◽

Shane C. Wright ◽

Kouki Kawakami ◽

Signe Mathiasen ◽

Joris Zhou ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Activation ◽

Receptor Internalization ◽

Protein Association ◽

Protein Activation ◽

Protein Receptor ◽

G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

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Pleiotropic Role for GRKs and b-Arrestins in Receptor Regulation

Physiology ◽

10.1152/physiologyonline.1997.12.4.145 ◽

1997 ◽

Vol 12 (4) ◽

pp. 145-152 ◽

Cited By ~ 1

Author(s):

SSG Ferguson ◽

J Zhang ◽

LS Barak ◽

MG Caron

Keyword(s):

Angiotensin Ii ◽

G Protein ◽

Intracellular Trafficking ◽

Coated Vesicle ◽

Receptor Regulation ◽

Receptor Desensitization ◽

G Protein Coupled Receptor ◽

Receptor Kinases ◽

Trafficking Molecules ◽

G Protein Coupled

G protein-coupled receptor kinases and arrestin proteins are well-characterized mediators of agonist-dependent G protein-coupled receptor desensitization. These proteins are now shown to play a dual role in receptor regulation by mediating both receptor uncoupling and sequestration, a process important for receptor resensitization. b-Arrestins bound to phosporylated b2-adrenergic and angiotensin II type 1A receptors act as intracellular trafficking molecules specifically targeting these receptors for dynamin-dependent clathrin-coated vesicle-mediated sequestration.

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Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2305 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2305-2324 ◽

Cited By ~ 70

Author(s):

Karen McConalogue ◽

Carlos U. Corvera ◽

Patrick D. Gamp ◽

Eileen F. Grady ◽

Nigel W. Bunnett

Keyword(s):

Plasma Membrane ◽

Substance P ◽

G Protein ◽

Neurokinin 1 Receptor ◽

Protein Receptor ◽

Early Endosomes ◽

Receptor Kinases ◽

Neurokinin 1 ◽

G Protein Coupled ◽

Neurotransmitter Substance

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Gαq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.

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