New Features on the Expression and Trafficking of mGluR1 Splice Variants Exposed by Two Novel Mutant Mouse Lines

Abstract

Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1β are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1β has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering.

Parvalbumin interneuron mGlu5receptors govern sex differences in prefrontal cortex physiology and binge drinking

“…mGlu receptor signaling is regulated at several levels that could vary by sex. One possibility is that Grm1 could display sex differences in splicing [73], leading to differences in subcellular protein expression and effector signaling [74,75]. Indeed, studies in hippocampal interneurons found that mGlu 1 and mGlu 5 receptors can activate distinct signaling pathways to regulate calcium mobilization [72], illustrating that mGlu 1 and mGlu 5 receptors could depolarize PV-INs through divergent mechanisms and these could also vary by sex.…”

mGluR5 Is Substitutable for mGluR1 in Cerebellar Purkinje Cells for Motor Coordination, Developmental Synapse Elimination, and Motor Learning

“…We previously generated mGluR1a-P/E-rescue mice expressing mGluR1a with a point mutation (proline (P) 1153 to glutamate (E)) in its Homer binding domain. We found that the P/E-mutant mGluR1a deficient for Homer binding did not significantly alter its distribution at perisynaptic sites of parallel fiber-PC synapses and rescued impaired motor coordination observed in mGluR1-KO mice [31]. Taken together with the lower amount of Homer binding with mGluR5 homodimers in mGluR5-rescue mice, the binding of Homer to mGluR1/5 may not contribute significantly to group I mGluR function in PCs.…”

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum

“…mGlu 1 β is a short alternatively spliced variant of the Grm1 gene in which the last 318 amino acid residues of the mGlu 1 α variant are replaced by 20 different residues [1]. Available evidence suggests that mGlu 1 α and mGlu 1 β variants heterodimerize both in vitro and in vivo [48][49][50] which affects receptor trafficking to the plasma membrane of Purkinje cell [46,47]. In line with these previous findings, we were able to identify specific mGlu 1 β peptides in five out of six experiments, despite the fact that the specific portion of the mGlu 1 β variant is quite short.…”