RESUMEN
The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Quinasa 5 del Receptor Acoplado a Proteína-G/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Calmodulina/genética , Calmodulina/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera , Especificidad por Sustrato , TermodinámicaRESUMEN
G protein-coupled receptor kinases (GRKs) phosphorylate the activated forms of G protein-coupled receptors (GPCRs), leading to receptor desensitization and internalization. In addition, GRKs can modify the activity of many non-GPCR-signaling pathways as well, controlling other cellular functions beyond that directly associated with a GPCR. In this report, we show that cervical cancer HeLa cells and breast cancer MDA MB 231 cells with reduced GRK5 expression display increased sensitivity to the apoptotic effects of paclitaxel (Taxol). This effect in cancer cells with low GRK5 levels could be because of blunted histone deacetylase 6 (HDAC6) activity that leads to an increase in α-tubulin acetylation levels, which augments paclitaxel sensitivity. We demonstrate that GRK5 and HDAC6 form a signaling complex in cells and in vitro. GRK5 phosphorylates HDAC6 at Ser-21 to promote its deacetylase activity. Therefore, the GRK5-HDAC6 interaction may contribute to paclitaxel resistance in cancer cells.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Paclitaxel/farmacología , Acetilación , Apoptosis/efectos de los fármacos , Biocatálisis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Docetaxel/farmacología , Femenino , Quinasa 3 del Receptor Acoplado a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Células HeLa , Histona Desacetilasa 6/metabolismo , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
During the development of heart failure (HF), the capacity for cardiomyocyte (CM) fatty acid oxidation (FAO) and ATP production is progressively diminished, contributing to pathologic cardiac hypertrophy and contractile dysfunction. Receptor-interacting protein 140 (RIP140, encoded by Nrip1) has been shown to function as a transcriptional corepressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibited increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and CM-specific RIP140-deficient (csNrip1-/-) mice were protected against the development of HF caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in CMs were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and fatty acid utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for the treatment of HF.
Asunto(s)
Insuficiencia Cardíaca , Proteína de Interacción con Receptores Nucleares 1 , Animales , Ratones , Cardiomegalia/metabolismo , Metabolismo Energético/genética , Ácidos Grasos/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Proteína de Interacción con Receptores Nucleares 1/genética , Proteína de Interacción con Receptores Nucleares 1/metabolismoRESUMEN
In the title compound, C23H38O5, the oxabicyclo-[2.2.1]heptane-2,3-dicarb-oxy-lic anhydride unit has a normal geometry and the tetra-decoxymethyl side chain is fully extended. In the crystal, mol-ecules are linked head-to-head by C-Hâ¯O hydrogen bonds, forming two-dimensional networks propagating along the a and c-axis directions.
RESUMEN
G protein-coupled receptors (GPCRs) interact with three protein families following agonist binding: heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs) and arrestins. GRK-mediated phosphorylation of GPCRs promotes arrestin binding to uncouple the receptor from G protein, a process called desensitization, and for many GPCRs, arrestin binding also promotes receptor endocytosis and intracellular signaling. Thus, GRKs play a central role in modulating GPCR signaling and localization. Here we review recent advances in this field which include additional insight into how GRKs target GPCRs and bias signaling, and the development of specific inhibitors to dissect GRK function in model systems.