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1.
J Chem Inf Model ; 61(9): 4190-4199, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34397210

RESUMO

Signaling bias is a feature of many G protein-coupled receptor (GPCR) targeting drugs with potential clinical implications. Whether it is therapeutically advantageous for a drug to be G protein biased or ß-arrestin biased depends on the context of the signaling pathway. Here, we explored GPCR ligands that exhibit biased signaling to gain insights into scaffolds and pharmacophores that lead to bias. More specifically, we considered BiasDB, a database containing information about GPCR biased ligands, and focused our analysis on ligands which show either a G protein or ß-arrestin bias. Five different machine learning models were trained on these ligands using 15 different sets of features. Molecular fragments which were important for training the models were analyzed. Two of these fragments (number of secondary amines and number of aromatic amines) were more prevalent in ß-arrestin biased ligands. After training a random forest model on HierS scaffolds, we found five scaffolds, which demonstrated G protein or ß-arrestin bias. We also conducted t-SNE clustering, observing correspondence between unsupervised and supervised machine learning methods. To increase the applicability of our work, we developed a web implementation of our models, which can predict bias based on user-provided SMILES, drug names, or PubChem CID. Our web implementation is available at: drugdiscovery.utep.edu/biasnet.


Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo
2.
Nat Commun ; 12(1): 4721, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354057

RESUMO

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) - a prototypical class A GPCR-activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and ß-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.


Assuntos
Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação/genética , Desenho de Fármacos , Humanos , Cadeias de Markov , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais , beta-Arrestinas/metabolismo
3.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361094

RESUMO

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine receptor (DR) heteromers are promising candidates for targeted therapy of neurological conditions such as Parkinson's disease since current treatments can have severe side effects. To facilitate development of such therapies, it is necessary to identify the various DR binding partners. We report here a new interaction partner for DRD2 and DRD3, the orphan receptor G protein-coupled receptor 143 (GPR143), an atypical GPCR that plays multiple roles in pigment cells and is expressed in several regions of the brain. We previously demonstrated that the DRD2/ DRD3 antagonist pimozide also modulates GPR143 activity. Using confocal microscopy and two FRET methods, we observed that the DRs and GPR143 colocalize and interact at intracellular membranes. Furthermore, co-expression of wildtype GPR143 resulted in a 57% and 67% decrease in DRD2 and DRD3 activity, respectively, as determined by ß-Arrestin recruitment assay. GPR143-DR dimerization may negatively modulate DR activity by changing affinity for dopamine or delaying delivery of the DRs to the plasma membrane.


Assuntos
Dopamina/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , beta-Arrestinas/metabolismo , Proteínas do Olho/genética , Humanos , Glicoproteínas de Membrana/genética , Mutação , Ligação Proteica , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/genética , Transdução de Sinais
4.
Nat Commun ; 12(1): 4357, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272386

RESUMO

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.


Assuntos
Citomegalovirus/metabolismo , Receptores de Quimiocinas/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Domínio Único/química , Esferoides Celulares/efeitos dos fármacos , Proteínas Virais/imunologia , Quimiocina CX3CL1/metabolismo , Cromatografia Líquida , Citomegalovirus/química , Células HEK293 , Humanos , Ligantes , Conformação Molecular , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Espectrometria de Massas em Tandem , beta-Arrestinas/metabolismo
5.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201646

RESUMO

The physiological and pathophysiological relevance of the angiotensin II type 1 (AT1) G protein-coupled receptor no longer needs to be proven in the cardiovascular system. The renin-angiotensin system and the AT1 receptor are the targets of several classes of therapeutics (such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers, ARBs) used as first-line treatments in cardiovascular diseases. The importance of AT1 in the regulation of the cerebrovascular system is also acknowledged. However, despite numerous beneficial effects in preclinical experiments, ARBs do not induce satisfactory curative results in clinical stroke studies. A better understanding of AT1 signaling and the development of biased AT1 agonists, able to selectively activate the ß-arrestin transduction pathway rather than the Gq pathway, have led to new therapeutic strategies to target detrimental effects of AT1 activation. In this paper, we review the involvement of AT1 in cerebrovascular diseases as well as recent advances in the understanding of its molecular dynamics and biased or non-biased signaling. We also describe why these alternative signaling pathways induced by ß-arrestin biased AT1 agonists could be considered as new therapeutic avenues for cerebrovascular diseases.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Lesões Encefálicas Traumáticas/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Humanos , Aneurisma Intracraniano/metabolismo , Terapia de Alvo Molecular/métodos , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , beta-Arrestinas/agonistas , beta-Arrestinas/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070125

RESUMO

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of ß-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused ß-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in ß-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced ß-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by ß-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and ß-arrestin-mediated responses.


Assuntos
Proteínas do Tecido Nervoso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Substância P/metabolismo , beta-Arrestinas/metabolismo , Substituição de Aminoácidos , Animais , Degranulação Celular , Linhagem Celular , Feminino , Humanos , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neuroimunomodulação/fisiologia , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Tirosina/química , beta-Arrestina 2/deficiência , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
7.
J Med Chem ; 64(12): 8710-8726, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34110150

RESUMO

Using a previously unexplored, efficient, and versatile multicomponent method, we herein report the rapid generation of novel potent and subtype-selective DRD2 biased partial agonists. This strategy exemplifies the search for diverse and previously unexplored moieties for the secondary/allosteric pharmacophore of the common phenyl-piperazine scaffold. The pharmacological characterization of the new compound series led to the identification of several ligands with excellent DRD2 affinity and subtype selectivity and remarkable functional selectivity for either the cAMP (22a and 24d) or the ß-arrestin (27a and 29c) signaling pathways. These results were further interpreted on the basis of molecular models of these ligands in complex with the recent DRD2 crystal structures, highlighting the critical role of the secondary/allosteric pharmacophore in modulating the functional selectivity profile.


Assuntos
Piperazinas/farmacologia , Receptores de Dopamina D2/agonistas , AMP Cíclico/metabolismo , Desenho de Fármacos , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/metabolismo , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , beta-Arrestinas/metabolismo
8.
J Biol Chem ; 297(1): 100881, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144038

RESUMO

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca2+, and ß-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca2+ and ß-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling, but unaffected agonist-stimulated ß-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure-function relationship studies of GPR17 signaling and metabolic disease.


Assuntos
Síndrome Metabólica/genética , Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo
9.
Mol Pharmacol ; 100(1): 7-18, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958480

RESUMO

Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, ß-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPγS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for ß-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of ß-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, ß-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , beta-Arrestinas/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Estrutura Molecular , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química
10.
Elife ; 102021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34013886

RESUMO

The prevailing model for the variety in drug responses is that different drugs stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand generates different GPCR active states based on the immediate environment of receptors is not known. Here we address this question using spatially resolved imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neuronal cells. We have shown that Golgi and surface pools of DOR both inhibit cAMP, but engage distinct conformational biosensors in response to the same ligand in rat neuroendocrine cells. Further, DOR recruits arrestins on the surface but not on the Golgi. Our results suggest that the local environment determines the active states of receptors for any given drug, allowing GPCRs to couple to different effectors at different subcellular locations.


Assuntos
Benzamidas/farmacologia , Membrana Celular/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piperazinas/farmacologia , Receptores Opioides delta/agonistas , Animais , Técnicas Biossensoriais , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Complexo de Golgi/metabolismo , Ligantes , Microscopia de Fluorescência , Neurônios/metabolismo , Células PC12 , Conformação Proteica , Ratos , Receptores Opioides delta/química , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Relação Estrutura-Atividade , beta-Arrestinas/metabolismo
11.
Commun Biol ; 4(1): 569, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980979

RESUMO

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.


Assuntos
Benzilaminas/farmacologia , Ciclamos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Receptores CXCR4/metabolismo , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacologia , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Benzilaminas/metabolismo , Butilaminas/metabolismo , Butilaminas/farmacologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Ciclamos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Feminino , Fator Estimulador de Colônias de Granulócitos , Células HEK293 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Preparações Farmacêuticas/metabolismo , Receptores CXCR3/efeitos dos fármacos , Receptores CXCR3/metabolismo , Receptores CXCR4/efeitos dos fármacos , beta-Arrestinas/efeitos dos fármacos , beta-Arrestinas/metabolismo
12.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806057

RESUMO

G protein-coupled receptor kinases (GRKs) are protein kinases that function in concert with arrestins in the regulation of a diverse class of G protein-coupled receptors (GPCRs) signaling. Although GRKs and arrestins are key participants in the regulation of GPCR cascades, the complex regulatory mechanisms of GRK expression, its alternation, and their function are not thoroughly understood. Several studies together with the work from our lab in recent years have revealed the critical role of these kinases in various physiological and pathophysiological processes, including cardiovascular biology, inflammation and immunity, neurodegeneration, thrombosis, and hemostasis. A comprehensive understanding of the mechanisms underlying functional interactions with multiple receptor proteins and how these interactions take part in the development of various pathobiological processes may give rise to novel diagnostic and therapeutic strategies. In this review, we summarize the current research linking the role of GRKs to various aspects of cell biology, pathology, and therapeutics, with a particular focus on thrombosis and hemostasis.


Assuntos
Arrestinas/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Quimiotaxia , Hemostasia , Humanos , Inflamação/imunologia , Fosforilação , Isoformas de Proteínas , Proteoma , Trombose , beta-Arrestinas/metabolismo
13.
J Biol Chem ; 296: 100503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33684444

RESUMO

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by ß-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or ß-arrestin-signaling pathways. However, nearly all screening techniques for measuring ß-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure ß-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to ß-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, ß-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of ß-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring ß-arrestin recruitment to diverse GPCR types in heterologous or native cells.


Assuntos
Membrana Celular/metabolismo , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Bioensaio/métodos , Células Cultivadas , Humanos , Ligantes , Ligação Proteica , Transdução de Sinais , beta-Arrestinas/química
14.
Circ Heart Fail ; 14(3): e007351, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33663236

RESUMO

BACKGROUND: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224. METHODS: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, ß-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output. RESULTS: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril. CONCLUSIONS: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.


Assuntos
Receptores de Apelina/agonistas , Débito Cardíaco/efeitos dos fármacos , Insuficiência Cardíaca/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Volume Sistólico/efeitos dos fármacos , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células CHO , Cricetulus , Cães , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Haplorrinos , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação , Ensaio Radioligante , Ratos , Trítio , Pressão Ventricular/efeitos dos fármacos , beta-Arrestinas/efeitos dos fármacos , beta-Arrestinas/metabolismo
15.
Science ; 371(6534)2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33479120

RESUMO

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and ß-arrestin adaptor proteins. G protein-mediated signaling and ß-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and ß-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:ß-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with ß-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:ß-arrestin signaling complexes.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Transdução de Sinais
16.
J Biol Chem ; 296: 100216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465377

RESUMO

For most G protein-coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein-coupled receptor kinase (GRK)-mediated phosphorylation, leading to ß-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:ß-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the ß-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and ß-arrestin binding. However, ß-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.


Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Miócitos de Músculo Liso/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Substituição de Aminoácidos , Brônquios/citologia , Brônquios/metabolismo , Cálcio/metabolismo , Difenidramina/farmacologia , Endossomos/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/química , Quinase 2 de Receptor Acoplado a Proteína G/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Mutação , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Taquifilaxia/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
17.
Biochim Biophys Acta Proteins Proteom ; 1869(4): 140603, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33421644

RESUMO

ß-arrestins bind active G protein-coupled receptors (GPCRs) and play a crucial role in receptor desensitization and internalization. The classical paradigm of arrestin function has been expanded with the identification of many non-receptor-binding partners, which indicated the multifunctional role of ß-arrestins in cellular functions. To elucidate the molecular mechanism of ß-arrestin-mediated signaling, the structural features of ß-arrestins were investigated using X-ray crystallography and cryogenic electron microscopy (cryo-EM). However, the intrinsic conformational flexibility of ß-arrestins hampers the elucidation of structural interactions between ß-arrestins and their binding partners using conventional structure determination tools. Therefore, structural information obtained using complementary structure analysis techniques would be necessary in combination with X-ray crystallography and cryo-EM data. In this review, we describe how ß-arrestins interact with their binding partners from a structural point of view, as elucidated by both traditional methods (X-ray crystallography and cryo-EM) and complementary structure analysis techniques.


Assuntos
beta-Arrestinas/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo
19.
Sci Rep ; 11(1): 1539, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452359

RESUMO

ß-Arrestins (ßArrs) are intracellular signal regulating proteins. Their expression level varies in some cancers and they have a significant impact on cancer cell function. In general, the significance of ßArrs in cancer research comes from studies examining GPCR signalling. Given the diversity of different GPCR signals in cancer cell regulation, contradictory results are inevitable regarding the role of ßArrs. Our approach examines the direct influence of ßArrs on cellular function and gene expression profiles by changing their expression levels in breast cancer cells, MDA-MB-231 and MDA-MB-468. Reducing expression of ßArr1 or ßArr2 tended to increase cell proliferation and invasion whereas increasing their expression levels inhibited them. The overexpression of ßArrs caused cell cycle S-phase arrest and differential expression of cell cycle genes, CDC45, BUB1, CCNB1, CCNB2, CDKN2C and reduced HER3, IGF-1R, and Snail. Regarding to the clinical relevance of our results, low expression levels of ßArr1 were inversely correlated with CDC45, BUB1, CCNB1, and CCNB2 genes compared to normal tissue samples while positively correlated with poorer prognosis in breast tumours. These results indicate that ßArr1 and ßArr2 are significantly involved in cell cycle and anticancer signalling pathways through their influence on cell cycle genes and HER3, IGF-1R, and Snail in TNBC cells.


Assuntos
Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , beta-Arrestinas/genética , Arrestinas/genética , Arrestinas/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Transdução de Sinais , beta-Arrestinas/metabolismo
20.
J Leukoc Biol ; 109(2): 373-376, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32480426

RESUMO

Atypical chemokine receptors (ACKRs) have emerged as important regulators or scavengers of homeostatic and inflammatory chemokines. Among these atypical receptors, ACKR4 is reported to bind the homeostatic chemokines CCL19, CCL21, CCL25 and CXCL13. In a recent study by Matti et al., the authors show that ACKR4 is also a receptor for CCL20, previously established to bind to CCR6 only. They provide convincing evidence that, just as for its other chemokine ligands, ACKR4 rapidly internalizes CCL20 both in vitro and in vivo. Independently of this discovery, we undertook a screening program aiming at reassessing the activity of the 43 human chemokines toward ACKR4 using a highly sensitive ß-arrestin recruitment assay. This systematic analysis confirmed CCL20 as a new agonist ligand for ACKR4 in addition to CCL19, CCL21, and CCL25. Furthermore, CCL22, which plays an important role in both homeostasis and inflammatory responses, and is known as a ligand for CCR4 and ACKR2 was found to also act as a potent partial agonist of ACKR4. In contrast, agonist activity of CXCL13 toward ACKR4 was disproved. This independent wide-range systematic study confirms the pairing of CCL20 with ACKR4 newly discovered by Matti and co-authors, and further refines the spectrum of chemokines activating ACKR4.


Assuntos
Quimiocina CCL20/metabolismo , Quimiocina CCL22/metabolismo , Quimiocina CXCL13/metabolismo , Receptores CCR/agonistas , Receptores CCR/metabolismo , Sequência de Aminoácidos , Quimiocina CCL22/química , Humanos , Ligantes , Filogenia , Ligação Proteica , beta-Arrestinas/metabolismo
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