IDENTIFICATION OF REGULATORY REGIONS CONTROLLING SECRETORY TRAFFICKING OF MGLUR6

Abstract

Vision is a process dependent on light detection by photoreceptors found in the retina. There are two kinds of photoreceptors, rods which are optimized for low-light vision and cones optimized for daylight and color vision. Rods are more sensitive and can detect a single photon and exclusively synapse with ON-pathway bipolar cells that detect increments of light. This is due to the kind of glutamate receptor expressed in the ON-bipolar cell, mGluR6, a class C GPCR that is negatively coupled to the cation channel TRPM1. In the dark mGluR6 is activated and, through a poorly understood mechanism, closes TRPM1, hyperpolarizing the ON-bipolar cells. Light onset reduces glutamate release and TRPM1 opens leading to ON-BC depolarization. Without mGluR6 the ON-BC signaling is not possible, and mutations in mGluR6 results in scotopic vision deficits. With the importance of mGluR6 to vision, very little is known about mechanisms that govern its secretory trafficking to the plasma membrane. Here we have identified the LBD to have important regions that not only influence surface expression but also regulates secretory trafficking pathway. Large deletions mutants of the LBD show increased surface expression in heterologous cells and displayed glycosylation characteristics indicative of a mix of both unconventional and conventional secretory trafficking. Shorter deletions in the LBD did not have increased surface expression but interestingly exclusively used unconventional protein secretion pathways, bypassing the Golgi. IP-MS was employed to find potential ER lumen resident proteins relevant to protein trafficking that is different between WT and deletion mutants and identified changes in stable binding to the ER lumen resident chaperone GRP78, that plays an important role in protein folding within the ER. Endogenous GRP78 binding was completely lost in the large deletion mutants but was present in CSNB mutants identified to be unconventionally trafficked proteins. The fluctuations in surface expression with mGluR6 LBD mutants implies and important regulatory role for the LBD in mGluR6 secretory trafficking.