RESUMEN
Within the intestine, the human G protein-coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrestin recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor-ß-arrestin interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G13 activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor-ß-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35-transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system.
Asunto(s)
Receptores Acoplados a Proteínas G , Regulación Alostérica , Cisteína/química , Disulfuros/química , Proteínas de Unión al GTP/química , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Ligandos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications.
Asunto(s)
Metabolismo Energético/fisiología , Fatiga/fisiopatología , Quinurenina/metabolismo , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Adaptación Fisiológica , Animales , Aspartato Aminotransferasas/metabolismo , Ácido Aspártico/metabolismo , Carbidopa/farmacología , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Metabolismo Energético/efectos de los fármacos , Glucólisis/fisiología , Malatos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Animales , Músculo Esquelético/fisiopatología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Condicionamiento Físico Animal/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transaminasas/antagonistas & inhibidores , Transaminasas/metabolismoRESUMEN
The role of tryptophan-kynurenine metabolism in psychiatric disease is well established, but remains less explored in peripheral tissues. Exercise training activates kynurenine biotransformation in skeletal muscle, which protects from neuroinflammation and leads to peripheral kynurenic acid accumulation. Here we show that kynurenic acid increases energy utilization by activating G protein-coupled receptor Gpr35, which stimulates lipid metabolism, thermogenic, and anti-inflammatory gene expression in adipose tissue. This suppresses weight gain in animals fed a high-fat diet and improves glucose tolerance. Kynurenic acid and Gpr35 enhance Pgc-1α1 expression and cellular respiration, and increase the levels of Rgs14 in adipocytes, which leads to enhanced beta-adrenergic receptor signaling. Conversely, genetic deletion of Gpr35 causes progressive weight gain and glucose intolerance, and sensitizes to the effects of high-fat diets. Finally, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. This work uncovers kynurenine metabolism as a pathway with therapeutic potential to control energy homeostasis.