GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation (original) (raw)
Related papers
Nature Communications, 2022
G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor–arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of β-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with β-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and β-arrestin mutants, we present evidence for differential receptor–β-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate t...
G protein coupled receptors as drug targets: the role of beta-arrestins
Endocrine, metabolic & immune disorders drug targets, 2008
G protein coupled receptors (GPCRs) are extremely important drug targets and the beta-arrestin intracellular scaffolding and adaptor proteins regulate major aspects of their pharmacology. beta-arrestin binding to activated, GPCR kinase (GRK)-phosphorylated receptors has the capacity to terminate G protein coupling, internalize the receptors into clathrin-coated vesicles and establish a secondary signaling complex independent of G protein signaling. These events appear to be differentially regulated by GRK phosphorylation, ubiquitination and potentially beta-arrestin oligomerization, which are likely to be highly receptor and cell-type dependent. The role of beta-arrestins in switching from G-protein dependent to independent signaling places them in a pivotal position to dictate the downstream effects of ligand binding. Consequently, we must appreciate the functioning of these molecules as we strive to discover and optimize new GPCR drug therapies for endocrine, metabolic and immune ...
GPCR Signaling Regulation: The Role of GRKs and Arrestins
Frontiers in Pharmacology
Every animal species expresses hundreds of different G protein-coupled receptors (GPCRs) that respond to a wide variety of external stimuli. GPCRs-driven signaling pathways are involved in pretty much every physiological function and in many pathologies. Therefore, GPCRs are targeted by about a third of clinically used drugs. The signaling of most GPCRs via G proteins is terminated by the phosphorylation of active receptor by specific kinases (GPCR kinases, or GRKs) and subsequent binding of arrestin proteins, that selectively recognize active phosphorylated receptors. In addition, GRKs and arrestins play a role in multiple signaling pathways in the cell, both GPCR-initiated and receptor-independent. Here we focus on the mechanisms of GRK-and arrestin-mediated regulation of GPCR signaling, which includes homologous desensitization and redirection of signaling to additional pathways by bound arrestins.
Dissecting the signaling features of the multi-protein complex GPCR/β-arrestin/ERK1/2
European Journal of Cell Biology, 2018
G protein-coupled receptors (GPCRs) have emerged as key biological entities that regulate a plethora of physiological processes and participate in the onset and development of many diseases. Moreover, these receptors are important targets of almost 25% of the current therapeutic drugs in the market. Upon agonist binding, GPCRs activate a great number of signaling pathways, resulting in important cellular events like gene transcription, survival, proliferation and differentiation. In order to activate such events, GPCRs interact with a variety of scaffold and molecular entities, particularly with G proteins, but also with β-arrestins and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, forming unique signaling modules. The aim of this review is to analyze the signaling features of the multi-protein complex GPCR-β-arrestin-ERK1/2, a unique signaling module that has received considerable attention from different research groups due to its molecular and physiological roles in diverse cellular contexts. Abbreviations: G protein-coupled receptors (GPCRs); Transmembrane (TM); Extracellular loops (ECLs); Intracellular loops (ICLs); G proteincoupled receptor kinases (GRKs); Mitogen activated protein kinases (MAPKs); Extracellular signal-regulated kinases 1 and 2 (ERK1/2); c-JUN Nterminal kinase (JNK); Receptor tyrosine kinases (RTKs); Epidermal growth factor (EGF); Platelet-derived growth factor (PDGF); m3-muscarinic receptor (M3-R); α2-adrenegic receptors (Α2-ARs); β2-adrenergic receptor (β2AR); Vasopressin-2 receptor (V2R); Protease-activated receptor-1 and-2 (PAR-1/2); Neurokinin-1 receptor (NK1R); Vasopressin-2 receptor phospho-peptide (V2Rpp); Receptor where the C-tail of β2AR is replaced by that of V2R (β2V2R); β1-adrenergic receptor (β1AR); Angiotensin-I A receptor (AT1AR); Angiotensin-II A receptor (AT12AR); Small interfering RNA (siRNA); Protein kinase C (PKC); RNA interference (RNAi); Angiotensin-I receptor (AT1R); Cyclic adenosine monophosphate (cAMP); Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9); Free fatty acid 4 receptor (FFA4); Follicle-stimulating hormone (FHS); Chemokine receptor 4 (CXCR4)
Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling
Molecular Systems Biology, 2012
Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through b-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT 1A R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on b-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT 1A R, and HEK293 cells expressing other 7TMRs.
Proceedings of the National Academy of Sciences of the United States of America, 2015
MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the...
Unraveling the molecular architecture of a G protein-coupled receptor/β-arrestin/Erk module complex
Scientific reports, 2015
β-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of β-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/β-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the β-arrestin/Raf1 and the β-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partner...
2003
It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that β2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 thus behaving as dual efficacy ligands. ERK1/2 activation by dual efficacy ligands was not affected by ADP-ribosylation of Gαi and could be observed in S49-cyc– cells lacking Gαs indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1/2 activation in a Gs/i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of β-arrestin and was abolished in mouse embryonic fi...
Targeting G protein–coupled receptor kinases to G protein–coupled receptors
Current Opinion in Endocrine and Metabolic Research, 2021
G protein-coupled receptors (GPCRs) interact with three protein families following agonist binding: heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs) and arrestins. GRK-mediated phosphorylation of GPCRs promotes arrestin binding to uncouple the receptor from G protein, a process called desensitization, and for many GPCRs, arrestin binding also promotes receptor endocytosis and intracellular signaling. Thus, GRKs play a central role in modulating GPCR signaling and localization. Here we review recent advances in this field which include additional insight into how GRKs target GPCRs and bias signaling, and the development of specific inhibitors to dissect GRK function in model systems.