Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay.

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Modificaciones en la distribución de neuropéptidos en el núcleo caudal del trigémino tras estimulación eléctrica del ganglio del trigérnino: modelo experimental de migraña / Changes in neuropeptide distribution in the caudal trigeminal nucleus following electrical stimulation of the trigeminal ganglion: an experimental migraine model

Con el fin de inducir algunas de las alteraciones que ocurren durante los ataques de migraña, hemos estimulado eléctricamente y unflateralmente el ganglio del trigémino de la rata, durante 5-30 minutos, para conocer en el núcleo caudal del trigémino las modifícaciones que aparecen en la distribución de los neuropéptidos sustancia P, neuroquinina A y péptido relacionado con el gen de la calcitonina. Una vez realizada la estimulación y la técnica inmunocitoquímica, observamos en secciones seriadas del núcleo caudal del trigémino ipsilateral una disminución signifícativa, con respecto al lado contralateral, de las terminaciones nerviosas inmunorreactivas que contienen a los neuropéptidos mencionados. Estas observaciones indican que durante los ataques de migraña, los tres neuropéptidos se liberan conjuntamente en el núcleo caudal del trigémino en donde actuarían a nivel de la primera sinapsis de la vía sensorial trigeminal como neurotransmisores y/o neuromoduladores de la información nociceptiva. (AU)