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1.
Nature ; 629(8011): 481-488, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38632411

RESUMO

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Receptores de Detecção de Cálcio , Humanos , Cálcio/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/química , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Sítios de Ligação , Estrutura Secundária de Proteína , Especificidade por Substrato
2.
Cell Signal ; 118: 111138, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38467243

RESUMO

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gßγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi3. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D2 receptor and influences inhibitory effect of Gαi3 on intracellular cAMP production triggered by D2 receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi3 binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Transdução de Sinais , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo
3.
Am J Physiol Cell Physiol ; 326(5): C1410-C1422, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38525541

RESUMO

Adipose dysfunction in lipodystrophic SEIPIN deficiency is associated with multiple metabolic disorders and increased risks of developing cardiovascular diseases, such as atherosclerosis, cardiac hypertrophy, and heart failure. Recently, adipose transplantation has been found to correct adipose dysfunction and metabolic disorders in lipodystrophic Seipin knockout mice; however, whether adipose transplantation could improve lipodystrophy-associated cardiovascular consequences is still unclear. Here, we aimed to explore the effects of adipose transplantation on lipodystrophy-associated metabolic cardiovascular diseases in Seipin knockout mice crossed into atherosclerosis-prone apolipoprotein E (Apoe) knockout background. At 2 months of age, lipodystrophic Seipin/Apoe double knockout mice and nonlipodystrophic Apoe knockout controls were subjected to adipose transplantation or sham operation. Seven months later, mice were euthanized. Our data showed that although adipose transplantation had no significant impact on endogenous adipose atrophy or gene expression, it remarkably increased plasma leptin but not adiponectin concentration in Seipin/Apoe double knockout mice. This led to significantly reduced hyperlipidemia, hepatic steatosis, and insulin resistance in Seipin/Apoe double knockout mice. Consequently, atherosclerosis burden, intraplaque macrophage infiltration, and aortic inflammatory gene expression were all attenuated in Seipin/Apoe double knockout mice with adipose transplantation. However, adipocyte morphology, macrophage infiltration, or fibrosis of the perivascular adipose tissue was not altered in Seipin/Apoe double knockout mice with adipose transplantation, followed by no significant improvement of vasoconstriction or relaxation. In conclusion, we demonstrate that adipose transplantation could alleviate lipodystrophy-associated metabolic disorders and atherosclerosis but has an almost null impact on perivascular adipose abnormality or vascular dysfunction in lipodystrophic Seipin/Apoe double knockout mice.NEW & NOTEWORTHY Adipose transplantation (AT) reverses multiply metabolic derangements in lipodystrophy, but whether it could improve lipodystrophy-related cardiovascular consequences is unknown. Here, using Seipin/Apoe double knockout mice as a lipodystrophy disease model, we showed that AT partially restored adipose functionality, which translated into significantly reduced atherosclerosis. However, AT was incapable of reversing perivascular adipose abnormality or vascular dysfunction. The current study provides preliminary experimental evidence on the therapeutic potential of AT on lipodystrophy-related metabolic cardiovascular diseases.


Assuntos
Tecido Adiposo , Aterosclerose , Subunidades gama da Proteína de Ligação ao GTP , Lipodistrofia , Camundongos Knockout , Animais , Camundongos , Tecido Adiposo/metabolismo , Tecido Adiposo/transplante , Apolipoproteínas E/genética , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Subunidades gama da Proteína de Ligação ao GTP/deficiência , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Resistência à Insulina , Leptina/sangue , Leptina/metabolismo , Lipodistrofia/metabolismo , Lipodistrofia/genética , Lipodistrofia/patologia , Camundongos Endogâmicos C57BL
4.
Plant Physiol Biochem ; 210: 108567, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38554538

RESUMO

Heterotrimeric G-proteins are key modulators of multiple signaling and developmental pathways in plants, in which they act as molecular switches to engage in transmitting various stimuli signals from outside into the cells. Substantial studies have identified G proteins as essential components of the organismal response to abiotic stress, leading to adaptation and survival in plants. Meanwhile, sugars are also well acknowledged key players in stress perception, signaling, and gene expression regulation. Connections between the two significant signaling pathways in stress response are of interest to a general audience in plant biology. In this article, advances unraveling a pivotal role of G proteins in the process of sugar signals outside the cells being translated into the operation of autophagy in cells during stress are reviewed. In addition, we have presented recent findings on G proteins regulating the response to drought, salt, alkali, cold, heat and other abiotic stresses. Perspectives on G-protein research are also provided in the end. Since G protein signaling regulates many agronomic traits, elucidation of detailed mechanism of the related pathways would provide useful insights for the breeding of abiotic stress resistant and high-yield crops.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Proteínas de Plantas , Transdução de Sinais , Estresse Fisiológico , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Açúcares/metabolismo , Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Fenômenos Fisiológicos Vegetais
5.
Dis Model Mech ; 17(6)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38454882

RESUMO

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP , Lipodistrofia Generalizada Congênita , Lipodistrofia , Animais , Humanos , Lipodistrofia Generalizada Congênita/genética , Lipodistrofia Generalizada Congênita/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Lipodistrofia/genética
6.
J Biol Chem ; 300(3): 105756, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38364891

RESUMO

Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Motivos de Aminoácidos , Membrana Celular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Humanos , Animais , Engenharia de Proteínas
7.
Stem Cells Dev ; 33(7-8): 177-188, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38386508

RESUMO

Seipin deficiency is an important cause of type 2 Berardinelli-Seip congenital dyslipidemia (BSCL2). BSCL2 is a severe lipodystrophy syndrome with lack of adipose tissue, hepatic steatosis, insulin resistance, and normal or higher bone mineral density. Bone marrow mesenchymal stem cells (BMSCs) are believed to maintain bone and fat homeostasis by differentiating into osteoblasts and adipocytes. We aimed to explore the role of seipin in the osteogenic/adipogenic differentiation balance of BMSCs. Seipin loxP/loxP mice are used to explore metabolic disorders caused by seipin gene mutations. Compared with wild-type mice, subcutaneous fat deficiency and ectopic fat accumulation were higher in seipin knockout mice. Microcomputed tomography of the tibia revealed the increased bone content in seipin knockout mice. We generated seipin-deficient BMSCs in vitro and revealed that lipogenic genes are downregulated and osteogenic genes are upregulated in seipin-deficient BMSCs. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) signaling is reduced in seipin-deficient BMSCs, while using the PPARγ activator increased the lipogenic differentiation and decreased osteogenic differentiation of seipin-deficient BMSCs. Our findings indicated that bone and lipid metabolism can be regulated by seipin through modulating the differentiation of mesenchymal stem cells. Thus, a new insight of seipin mutations in lipid metabolism disorders was revealed, providing a prospective strategy for MSC transplantation-based treatment of BSCL2.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP , Células-Tronco Mesenquimais , Animais , Camundongos , Diferenciação Celular/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Knockout , Osteogênese/genética , PPAR gama/genética , PPAR gama/metabolismo , Microtomografia por Raio-X
8.
Nature ; 626(8001): 1141-1148, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38326620

RESUMO

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Receptores de Detecção de Cálcio , Humanos , Regulação Alostérica/efeitos dos fármacos , Cinacalcete/farmacologia , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ligantes , Lipídeos , Nanoestruturas/química , Poliaminas/metabolismo , Conformação Proteica/efeitos dos fármacos , Receptores de Detecção de Cálcio/química , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/ultraestrutura , Especificidade por Substrato , Triptofano/metabolismo , Cálcio/metabolismo
9.
Pharmacol Ther ; 255: 108589, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38295906

RESUMO

The original paradigm of classical - also referred to as canonical - cellular signal transduction of heterotrimeric G proteins (G protein) is defined by a hierarchical, orthograde interaction of three players: the agonist-activated G protein-coupled receptor (GPCR), which activates the transducing G protein, that in turn regulates its intracellular effectors. This receptor-transducer-effector concept was extended by the identification of regulators and adapters such as the regulators of G protein signaling (RGS), receptor kinases like ßARK, or GPCR-interacting arrestin adapters that are integrated into this canonical signaling process at different levels to enable fine-tuning. Finally, the identification of atypical signaling mechanisms of classical regulators, together with the discovery of novel modulators, added a new and fascinating dimension to the cellular G protein signal transduction. This heterogeneous group of accessory G protein modulators was coined "activators of G protein signaling" (AGS) proteins and plays distinct roles in canonical and non-canonical G protein signaling pathways. AGS proteins contribute to the control of essential cellular functions such as cell development and division, intracellular transport processes, secretion, autophagy or cell movements. As such, they are involved in numerous biological processes that are crucial for diseases, like diabetes mellitus, cancer, and stroke, which represent major health burdens. Although the identification of a large number of non-canonical G protein signaling pathways has broadened the spectrum of this cellular communication system, their underlying mechanisms, functions, and biological effects are poorly understood. In this review, we highlight and discuss atypical G protein-dependent signaling mechanisms with a focus on inhibitory G proteins (Gi) involved in canonical and non-canonical signal transduction, review recent developments and open questions, address the potential of new approaches for targeted pharmacological interventions.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Transdução de Sinais , Humanos , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
10.
Plant J ; 117(2): 616-631, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37910396

RESUMO

The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.


Assuntos
Aminoácidos , Proteínas de Arabidopsis , Arabidopsis , Ascomicetos , Proteínas Heterotriméricas de Ligação ao GTP , Indenos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Doenças das Plantas/genética
11.
Structure ; 32(1): 47-59.e7, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-37989308

RESUMO

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Transdução de Sinais , Transdução de Sinais/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ligação Proteica , Neurotransmissores
12.
Mol Cell Proteomics ; 23(2): 100705, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38135118

RESUMO

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas RGS , Arabidopsis/metabolismo , Fosforilação , Proteínas de Arabidopsis/metabolismo , Proteoma/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Proteínas RGS/metabolismo , Transdução de Sinais , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Flagelina/farmacologia , Flagelina/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
13.
PLoS Genet ; 19(11): e1011015, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37910589

RESUMO

Heterotrimeric G (αßγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cílios , Fatores de Troca do Nucleotídeo Guanina , Proteínas Heterotriméricas de Ligação ao GTP , Morfogênese , Animais , Humanos , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cílios/genética , Cílios/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Morfogênese/genética , Nucleotídeos/metabolismo , Células Receptoras Sensoriais/metabolismo
14.
Nature ; 624(7992): 663-671, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37935377

RESUMO

Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous ß-phenylethylamine (ß-PEA)1 as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1-G-protein complexes bound to methamphetamine and ß-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.


Assuntos
Metanfetamina , Fenetilaminas , Receptores Acoplados a Proteínas G , Humanos , Ligantes , Metanfetamina/metabolismo , Doenças do Sistema Nervoso/metabolismo , Fenetilaminas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Polifarmacologia , Ligação de Hidrogênio
15.
Planta ; 258(5): 101, 2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37847414

RESUMO

MAIN CONCLUSION: A comprehensive understanding of nitrogen signaling cascades involving heterotrimeric G-proteins and their putative receptors can assist in the production of nitrogen-efficient plants. Plants are immobile in nature, so they must endure abiotic stresses including nutrient stress. Plant development and agricultural productivity are frequently constrained by the restricted availability of nitrogen in the soil. Non-legume plants acquire nitrogen from the soil through root membrane-bound transporters. In depleted soil nitrogen conditions, legumes are naturally conditioned to fix atmospheric nitrogen with the aid of nodulation elicited by nitrogen-fixing bacteria. Moreover, apart from the symbiotic nitrogen fixation process, nitrogen uptake from the soil can also be a significant secondary source to satisfy the nitrogen requirements of legumes. Heterotrimeric G-proteins function as molecular switches to help plant cells relay diverse stimuli emanating from external stress conditions. They are comprised of Gα, Gß and Gγ subunits, which cooperate with several downstream effectors to regulate multiple plant signaling events. In the present review, we concentrate on signaling mechanisms that regulate plant nitrogen nutrition. Our review highlights the potential of heterotrimeric G-proteins, together with their putative receptors, to assist the legume root nodule symbiosis (RNS) cascade, particularly during calcium spiking and nodulation. Additionally, the functions of heterotrimeric G-proteins in nitrogen acquisition by plant roots as well as in improving nitrogen use efficiency (NUE) have also been discussed. Future research oriented towards heterotrimeric G-proteins through genome editing tools can be a game changer in the enhancement of the nitrogen fixation process. This will foster the precise manipulation and production of plants to ensure global food security in an era of climate change by enhancing crop productivity and minimizing reliance on external inputs.


Assuntos
Fabaceae , Proteínas Heterotriméricas de Ligação ao GTP , Rhizobium , Nódulos Radiculares de Plantas/genética , Nodulação/fisiologia , Nitrogênio/metabolismo , Fixação de Nitrogênio , Fabaceae/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Simbiose/fisiologia , Plantas/metabolismo , Verduras/metabolismo , Solo , Rhizobium/fisiologia
16.
J Biol Chem ; 299(11): 105269, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37739036

RESUMO

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Prenilação de Proteína , Humanos , Motivos de Aminoácidos , Resistência a Medicamentos/genética , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Modelos Moleculares , Mutação , Prenilação de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
17.
Trends Plant Sci ; 28(12): 1406-1421, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37625950

RESUMO

Heterotrimeric G-protein-mediated signaling is a key mechanism to transduce a multitude of endogenous and environmental signals in diverse organisms. The scope and expectations of plant G-protein research were set by pioneering work in metazoans. Given the similarity of the core constituents, G-protein-signaling mechanisms were presumed to be universally conserved. However, because of the enormous diversity of survival strategies and endless forms among eukaryotes, the signal, its interpretation, and responses vary even among different plant groups. Earlier G-protein research in arabidopsis (Arabidopsis thaliana) has emphasized its divergence from Metazoa. Here, we compare recent evidence from diverse plant lineages with the available arabidopsis G-protein model and discuss the conserved and novel protein components, signaling mechanisms, and response regulation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Heterotriméricas de Ligação ao GTP , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transdução de Sinais , Plantas/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas de Plantas/metabolismo
18.
Nature ; 620(7975): 904-910, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37558880

RESUMO

Arrestins have pivotal roles in regulating G protein-coupled receptor (GPCR) signalling by desensitizing G protein activation and mediating receptor internalization1,2. It has been proposed that the arrestin binds to the receptor in two different conformations, 'tail' and 'core', which were suggested to govern distinct processes of receptor signalling and trafficking3,4. However, little structural information is available for the tail engagement of the arrestins. Here we report two structures of the glucagon receptor (GCGR) bound to ß-arrestin 1 (ßarr1) in glucagon-bound and ligand-free states. These structures reveal a receptor tail-engaged binding mode of ßarr1 with many unique features, to our knowledge, not previously observed. Helix VIII, instead of the receptor core, has a major role in accommodating ßarr1 by forming extensive interactions with the central crest of ßarr1. The tail-binding pose is further defined by a close proximity between the ßarr1 C-edge and the receptor helical bundle, and stabilized by a phosphoinositide derivative that bridges ßarr1 with helices I and VIII of GCGR. Lacking any contact with the arrestin, the receptor core is in an inactive state and loosely binds to glucagon. Further functional studies suggest that the tail conformation of GCGR-ßarr governs ßarr recruitment at the plasma membrane and endocytosis of GCGR, and provides a molecular basis for the receptor forming a super-complex simultaneously with G protein and ßarr to promote sustained signalling within endosomes. These findings extend our knowledge about the arrestin-mediated modulation of GPCR functionalities.


Assuntos
Receptores de Glucagon , beta-Arrestina 1 , beta-Arrestina 1/química , beta-Arrestina 1/metabolismo , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Glucagon/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ligantes , Fosfatidilinositóis/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Ligação Proteica
19.
PLoS One ; 18(7): e0289098, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37490473

RESUMO

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.


Assuntos
Neoplasias da Mama , Proteínas Heterotriméricas de Ligação ao GTP , Receptores Nicotínicos , Humanos , Feminino , Nicotina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Sinalização do Cálcio , Receptores Nicotínicos/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proliferação de Células , Colina/farmacologia , Cálcio/metabolismo
20.
Int J Mol Sci ; 24(13)2023 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-37446209

RESUMO

Heterotrimeric G protein-mediated signaling plays a vital role in physiological and developmental processes in eukaryotes. On the other hand, because of the absence of a G protein-coupled receptor and self-activating mechanism of the Gα subunit, plants appear to have different regulatory mechanisms, which remain to be elucidated, compared to canonical G protein signaling established in animals. Here we report that Arabidopsis heterotrimeric G protein subunits, such as Gα (GPA1) and Gß (AGB1), regulate plant growth under stress conditions through the analysis of heterotrimeric G protein mutants. Flg22-mediated growth inhibition in wild-type roots was found to be caused by a defect in the elongation zone, which was partially blocked in agb1-2 but not gpa1-4. These results suggest that AGB1 may negatively regulate plant growth under biotic stress conditions. In addition, GPA1 and AGB1 exhibited genetically opposite effects on FCA-mediated growth inhibition under heat stress conditions. Therefore, these results suggest that plant G protein signaling is probably related to stress-mediated growth regulation for developmental plasticity in response to biotic and abiotic stress conditions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Subunidades beta da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas de Arabidopsis/metabolismo , Mutação , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica de Plantas , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo
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