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Yan Cao, Kirill A Martemyanov; Molecular determinants for synaptic targeting of mGluR6 in retinal ON-bipolar neurons. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4529.
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G protein-coupled receptor mGluR6 is the key molecule mediating the synaptic transmission in ON-bipolar cells (ON-BC) at the first visual synapse with photoreceptors. The localization of mGluR6 in ON-BC is restricted to the postsynaptic compartment at the dendritic tips. However, when expressed in transfected cells, mGluR6 is localized throughout the entire plasma membrane. This suggests that mGluR6 localization in ON-BC is determined by an active targeting mechanism. Furthermore, elimination of mGluR6 not only abolishes depolarizing response of ON-BC, but also impairs postsynaptic accumulation of other signaling components including RGS proteins and an effector channel TRPM1. While the spatial restriction of signaling at synapse is thought to be an important contributor to the timely responses of ON-BC, virtually nothing is known about how this organization is achieved. In this study, we investigated molecular determinants in mGluR6 that ensure its selective postsynaptic delivery by analyzing subcellular localization of various mGluR6 mutant constructs following their in vivo expression in mouse retinas.
Mouse retinas were electroporated with GFP tagged constructs driven by minimal mGluR6/SV40 hybrid promoter after subretinal microinjections at P0 and harvested at P21. The subcellular localization of the constructs was analyzed by confocal microscopy following their immunohistochemical detection.
We first investigated whether mGluR6 has unique targeting elements not present in other mGluRs. We ectopically expressed GFP tagged mGluR8 that shares substantial amino acid similarity and G protein coupling specificity with mGluR6. In other neurons, mGluR8 is documented to be localized at axonal terminals. However, when expressed in ON-BC mGluR8 accumulated at the dendritic tips, similarly to mGluR6. We further performed mutagenesis studies with mGluR6. Deletion of the C terminus didn’t impair postsynaptic targeting of mGluR6. However, deletion of ligand binding or cysteine-rich domains resulted in marked destabilization of mGluR6 complicating examination of their subcellular localization.
mGluR6 and mGluR8, two different metabotropic glutamate receptors are both targeted to ON-BC dendritic tips. This points to the absence of a unique postsynaptic targeting determinant specific to mGluR6. Instead targeting of mGluRs in ON-BC may rely on recognition of common structural features.
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