Investigação do interactoma do receptor metabotrópico de glutamato do tipo 5 (MGLUR5): enfoque no fator de crescimento neuronal 1 (NEGR1)

Abstract

Glutamate is the main excitatory neurotransmitter in the brain and exerts its effects through two classes of receptors, ionotropic and metabotropic. mGluR5 acts through the Gαq/11-βγ protein, leading to the activation of phospholipase C (PLC) that hydrolyzes phosphotidylinositol 4,5-bisphosphate (PIP2) in 1,2, -diacilglycerol (DAG) and inositol 1,4,5 triphosphate (IP3), leading to the release of Ca2+ from intracellular stores and activation of protein kinase C (PKC). In addition to the classic G protein pathway, mGluR5 interacts with other proteins through its intracellular and extracellular portion, leading to the activation of various cell signalling pathways. These interactions are important for numerous regulatory cell signaling pathways involved in diseases that affect the brain. By employing nanoscale liquid chromatography coupled with tandem mass spectrometry, we identified that extracellular cell adhesion molecules (CAMs) belonging to the Immunoglobulin superfamily co-immunoprecipitate with endogenous mGluR5 in hippocampal cell lysates. Furthermore, co-immunoprecipitation assays confirmed that one of these CAMs, neuronal growth regulator 1 (NEGR1), interacts with mGluR5 in cell lysates from hippocampus and cortex of C57/Bl6 mice and from primary neuronal culture. NEGR1 is an adhesion molecule, attached to the plasma membrane through a Glycosylphosphatidylinositol (GPI) anchor, that can be shed to the extracellular medium, playing an important role in neuronal development and synaptogenesis. Furthermore, our results show that NEGR1 is increased in the plasma membrane of mGluR5-/- primary neurons. However, both intracellular and secreted levels of NEGR1 are not significantly altered due to mGluR5 ablation. Interestingly, by exploring the effects of NEGR1 on intracellular mGluR5 signaling, we showed that mGluR5 agonist (DHPG)-induced kinase (ERK1/2) activation is reduced due to NEGR1silencing by lentiviral vectors, indicating that NEGR1 may participate in DHPG-induced ERK1/2 activation. However, the analysis of genes regulated by mGluR5 showed that treatment with DHPG leads to a significant increase in ARC/ARG3.1 expression, although this was observed both in neurons expressing endogenous levels of NEGR1 and in NEGR1 knockdown neurons. Furthermore, when we analyzed the basal expression levels of these genes, we observed that the knockdown of NEGR1 significantly decreased the transcripts of both BDNF and ARC/ARG3.1. Furthermore, preliminary data indicates that treatment with recombinant NEGR1 may be sufficient to elevate ARC/ARG3.1 levels in the absence of DHPG. Together, these data indicate that NEGR1 interacts with mGluR5 and plays an important role in the receptor’s cell signaling.