ERK1/2 signalling coupled to 5-HT4 receptor and its regulation by GRK5

Les récepteurs couplés aux protéines G (RCPG) peuvent activer des voies de signalisation indépendantes des protéines G. Cependant, la régulation de ces voies, et en particulier leur désensibilisation, est peu connue. Le récepteur de la sérotonine de type 4 (R-5-HT4) est un RCPG exprimé dans le cerveau et les organes périphériques. Il est impliqué dans des fonctions physiologiques importantes comme la mémoire, l'apprentissage, la prise de nourriture, le contrôle respiratoire et la mobilité gastro-intestinale. Le R-5-HT4 est couplé à la protéine Gs. De plus, il active la voie Src/ERK1/2, indépendamment des protéines G et des β-arrestines.Nous avons montré que GRK5, physiquement associé à la région C-terminale (C-ter) du R-5-HT4 inhibait la voie Src/ERK1/2 couplée au récepteur, mais pas la voie Gs. Ce résultat a été observé dans la lignée de cellules HEK-293 mais aussi dans des neurones de collicules en culture. Cette inhibition nécessite deux séquences d'évènements : l'association de la β-arrestine1 à une région riche en sérines et thréonines, localisée dans le domaine C-ter du récepteur et la phosphorylation par GRK5, de la β-arrestine1 (en sérine 412) liée au récepteur. La β-arrestine1 phosphorylée empêche l'activation de Src, constitutivement liée au récepteur, nécessaire à l'activation d'ERK1/2. Ceci constitue la première démonstration que la phosphorylation d'une β-arrestine par une GRK régule la signalisation indépendante des protéines G. En plus de ces résultats, nous avons démontré que l'activation d'ERK1/2 par le R-5-HT4, indépendante des β-arrestines, implique la libération d'un ligand induite par une métalloprotéase, conduisant à la transactivation d'un autre récepteur. Par une approche protéomique, nous avons également identifiés plusieurs partenaires potentiels du R-5-HT4. L'étude de ces partenaires pourrait apporter un éclairage supplémentaire sur les voies de signalisation du récepteur et leur régulation.G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized.The 5-Hydroxytryptamine 4 receptor (5-HT4R) is a GPCR widely expressed in the brain and at the periphery. It is implicated in important physiological functions such as memory, cognition, feeding, respiratory control and gastrointestinal motility. 5-HT4R couples to the Gs/cAMP/PKA pathway. Moreover, this receptor can activate a Src/ERK pathway independently of both G proteins and β-arrestins.Here, we show that the G protein-independent 5-HT4R-operated Src/ERK pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor C-terminus, in both HEK-293 cells and colliculi neurons. This inhibition requires two sequences of events: the association of β-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation by GRK5 of β-arrestin1 (at Ser 412) bound to the receptor. Phosphorylated β-arrestin1 prevents in turn activation of Src constitutively bound to 5-HT4R, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin phosphorylation by a GRK regulates G protein-independent signalling.In addition to these results, we also demonstrated that the β-arrestin-independent activation of ERK1/2 by the 5-HT4R involves a metalloprotease-dependant ectodomain shedding and transactivation of another receptor. By a proteomic approach, we also identified several potential partners of the 5-HT4R. Study of these proteins may provide a better understanding of 5-HT4R signalling and his regulation

β-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT 4 receptor signalling

International audienceG protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT4 receptor (5‐HT4R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser412) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT4Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling

Excess risk of urinary tract cancers in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study

IF 6.32International audienceBackgroundThe risk of urinary tract cancers, including kidney and bladder cancers, was increased in transplant recipients receiving thiopurines.AimTo assess the risk of urinary tract cancers in patients with inflammatory bowel disease (IBD) receiving thiopurines in the CESAME observational cohort.MethodsBetween May 2004 and June 2005, 19 486 patients with IBD, 30.1% of whom were receiving thiopurines, were enrolled. Median follow‐up was 35 months (IQR: 29–40).ResultsTen and six patients developed respectively kidney and bladder cancer. The incidence rates of urinary tract cancer were 0.48/1000 patient‐years in patients receiving thiopurines (95% CI: 0.21–0.95), 0.10/1000 patient‐years in patients who discontinued thiopurines (95% CI: 0.00–0.56) and 0.30/1000 patient‐years in patients never treated with thiopurines (95% CI: 0.12–0.62) at entry. The standardised incidence ratio of urinary tract cancer was 3.40 (95% CI: 1.47–6.71, P = 0.006) in patients receiving thiopurines, 0.64 (95% CI: 0.01–3.56, P = 0.92) in patients previously exposed to thiopurines and 1.17 (95% CI: 0.47–12.42, P = 0.78) in patients never treated with thiopurines. The multivariate‐adjusted hazard ratio (HR) of urinary tract cancer between patients receiving thiopurines and those not receiving thiopurines was 2.82 (95% CI: 1.04–7.68, P = 0.04). Other significant risk factors were male gender (HR: 3.98, 95% CI: 1.12–14.10, P = 0.03) and increasing age (HR after 65 years (ref <50): 13.26, 95% CI: 3.52–50.03, P = 0.0001).ConclusionPatients with IBD receiving thiopurines have an increased risk of urinary tract cancers. Clinically relevant excess risk is observed in older men