RESUMO
During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that ß-arrestin1 and ß-arrestin2 (ß-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that ß-arrestins are expressed in rat commissural neurons. We also found that Smo, ß-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on ß-arrestins. ß-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that ß-arrestins are required to activate Src kinase downstream of Shh. ß-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative ß-arrestins, ß-arrestin1 V53D which blocks the internalization of Smo and ß-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative ß-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that ß-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.
Assuntos
Orientação de Axônios , Proteínas Hedgehog , beta-Arrestinas , Animais , Proteínas Hedgehog/metabolismo , Ratos , Orientação de Axônios/fisiologia , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Arrestinas/genética , Feminino , Axônios/fisiologia , Axônios/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Quinases da Família src/metabolismo , Masculino , Medula Espinal/metabolismo , Medula Espinal/embriologia , Medula Espinal/citologia , Embrião de Galinha , HumanosRESUMO
Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.
Assuntos
Arrestinas , Fibras Musculares Esqueléticas , Transtornos Musculares Atróficos , Animais , Camundongos , Envelhecimento/genética , Arrestinas/genética , Arrestinas/metabolismo , Músculo Esquelético , Atrofia Muscular/genética , RNA Mensageiro/genéticaRESUMO
The first member of the arrestin family, visual arrestin-1, was discovered in the late 1970s. Later, the other three mammalian subtypes were identified and cloned. The first described function was regulation of G protein-coupled receptor (GPCR) signaling: arrestins bind active phosphorylated GPCRs, blocking their coupling to G proteins. It was later discovered that receptor-bound and free arrestins interact with numerous proteins, regulating GPCR trafficking and various signaling pathways, including those that determine cell fate. Arrestins have no enzymatic activity; they function by organizing multi-protein complexes and localizing their interaction partners to particular cellular compartments. Today we understand the molecular mechanism of arrestin interactions with GPCRs better than the mechanisms underlying other functions. However, even limited knowledge enabled the construction of signaling-biased arrestin mutants and extraction of biologically active monofunctional peptides from these multifunctional proteins. Manipulation of cellular signaling with arrestin-based tools has research and likely therapeutic potential: re-engineered proteins and their parts can produce effects that conventional small-molecule drugs cannot.
Assuntos
Arrestinas , Transdução de Sinais , Humanos , Animais , Arrestinas/metabolismo , Arrestinas/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Ligação Proteica , FosforilaçãoRESUMO
Purpose: Variants in the ARR3 gene have been linked to early-onset high myopia (eoHM) with a unique X-linked female-limited inheritance. However, the clinical validity of this gene-disease association has not been systematically evaluated. Methods: We identified two Chinese families with novel ARR3 splicing variants associated with eoHM. Minigene constructs were generated to assess the effects of the variants on splicing. We integrated previous evidence to curate the clinical validity of ARR3 and eoHM using the ClinGen framework. Results: The variants c.39+1G>A and c.100+4A>G were identified in the two families. Minigene analysis showed both variants resulted in abnormal splicing and introduction of premature termination codons. Based on genetic and experimental evidence, the ARR3-eoHM relationship was classified as "definitive." Conclusions: Our study identified two novel splicing variants of the ARR3 gene linked to eoHM and confirmed their functional validity via minigene assay. This research expanded the mutational spectrum of ARR3 and confirmed the minigene assay technique as an effective tool for understanding variant effects on splicing mechanisms.
Assuntos
Arrestinas , Miopia , Splicing de RNA , Feminino , Humanos , Mutação , Miopia/genética , Splicing de RNA/genética , Arrestinas/genética , População do Leste Asiático/genéticaRESUMO
Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.
Assuntos
Linfoma , Neoplasias , Animais , Camundongos , Arrestinas/genética , Arrestinas/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias/genéticaRESUMO
GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (ß-arrestin 1) and Arrestin 3 (ß-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.
Assuntos
Arrestinas , Receptores Acoplados a Proteínas G , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , beta-Arrestina 2/metabolismo , Hormônio do Crescimento , Clatrina/metabolismo , EndocitoseRESUMO
The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require ß-arrestins. While differences in GLP-1R trafficking and ß-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and ß-arrestin recruitment profiles using cells where endogenous ß-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical ß-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R ß-arrestin 1 and ß-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with ß-arrestin 1 recruitment more sensitive to GRK knockout than ß-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated ß-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.
Assuntos
Diabetes Mellitus Tipo 2 , Quinases de Receptores Acoplados a Proteína G , Humanos , Arrestinas/genética , Arrestinas/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo , beta-Arrestinas/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células HEK293 , Fosforilação , Receptores Acoplados a Proteínas G/metabolismoRESUMO
CXC motif chemokine 12 (CXCL12) promotes metastasis of several tumors by affecting cell migration and invasion via its receptors, CXC chemokine receptor type (CXCR)4 and CXCR7. Current therapeutic approaches focus on the selective inactivation of either CXCR4 or CXCR7 in patients with cancer. Alternative strategies may emerge from the analysis of downstream events that mediate the migratory effects of CXCL12 in cancer cells. While CXCR4 activates cell signaling through both G proteins and arrestins, CXCR7 is believed to preferentially signal through arrestins. The present study analyzed the CXCL12dependent chemotaxis of A549, C33A, DLD1, MDAMB231 and PC3 cells, in which either the activity of G proteins, EGFR or Src kinase was inhibited pharmacologically or the expression of arrestins was inhibited by RNA interference. The results demonstrated that CXCL12induced migration of A549, C33A, DLD1, MDAMB231 and PC3 cells was attenuated by the Gαi/oinhibitor pertussis toxin (PTX), but was unaffected by small interfering RNAmediated gene silencing of ßarrestin1/2. In particular, the sensitivity of DLD1 migration to PTX was unexpected, as it is solely dependent on the nonclassical chemokine receptor, CXCR7. Furthermore, chemotactic responses to CXCL12 were additionally prevented by inhibiting EGFR activity via AG1478 and Src kinase activity via Src inhibitor1. In conclusion, the results of the present study suggest that G protein and Srcdependent transactivation of EGFR is a common mechanism through which CXCL12bound CXCR4 and/or CXCR7 control cancer cell migration and metastasis. These findings highlight EGFR as a potential therapeutic target that interferes with CXCL12induced cancer expansion.
Assuntos
Neoplasias , Receptores CXCR , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Ativação Transcricional , Receptores CXCR/genética , Receptores CXCR/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Movimento Celular , Arrestinas/genética , Arrestinas/metabolismo , Arrestinas/farmacologia , Quinases da Família src/genética , Quinases da Família src/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismoRESUMO
In rodents with unilateral ablation of neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA induces a progressive increase of behavioral responses, a process known as behavioral sensitization. This sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of these mice, we find that the restoration of arrestin-3 fully rescues behavioral sensitization, whereas its mutant defective in c-Jun N-terminal kinase (JNK) activation does not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in direct pathway striatal neurons, also fully rescues sensitization, whereas an inactive homologous arrestin-2-derived peptide does not. Behavioral rescue is accompanied by the restoration of JNK3 activity, as reflected by JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-assisted JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization upon dopamine depletion and chronic L-DOPA treatment.
Assuntos
Arrestinas , Comportamento Animal , Dopamina , Camundongos Knockout , Proteína Quinase 10 Ativada por Mitógeno , Animais , Humanos , Camundongos , Arrestinas/metabolismo , Arrestinas/genética , Comportamento Animal/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Levodopa/farmacologia , Camundongos Endogâmicos C57BL , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/genética , Fosforilação/efeitos dos fármacosRESUMO
Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Results: Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.