Night blindness-associated mutations in the ligand-binding, cysteine-rich, and intracellular domains of the metabotropic glutamate receptor 6 abolish protein trafficking

Abstract

Mutations in the GRM6 gene, which encodes the metabotropic glutamate receptor 6 (mGluR6), lead to autosomal recessive congenital stationary night blindness (CSNB), which is characterized by loss of night vision due to a defect in signal transmission from photoreceptor to the adjacent ON-bipolar cells in the retina. So far, the sequence variations that have been described in six different families include nonsense, frameshift, and missense mutations. Here we investigated the impact of missense mutations in the ligand-binding domain, a conserved cysteine-rich domain, and the intracellular domain on the localization of the protein. We visualized and discriminated between surface and intracellular protein. Here we demonstrate that the wild-type (wt) protein localizes to the cell surface, and to endoplasmic reticulum (ER) and Golgi compartments. This also holds true for a mGluR6 variant containing a polymorphic, nondisease-associated amino acid exchange in the ligand-binding domain. In contrast, all disease-associated missense mutations lead to retention of the protein in the ER, while dimerization seems not to be affected. This is the first report that shows that CSNB-associated mutations in three different domains of mGluR6 abolish proper protein trafficking. We propose that the ligand-binding and the poorly characterized cysteine-rich domains, in addition to the intracellular domains, have a pivotal role in correct trafficking of metabotropic glutamate receptors to the cell surface.

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

“…A number of mutations in the TMD of class C GPCRs which result in either a gain or a loss of function have been reported, either through site-directed mutagenesis (28) or through the analysis of human gene sequences (29)(30)(31)(32). For example, both gain-and loss-of-function mutations identified in the calciumsensing receptor gene are responsible for autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively (33).…”

Congenital stationary night blindness with hypoplastic discs, negative electroretinogram and thinning of the inner nuclear layer

“…Upon light exposure, the cessation of glutamate release from the photoreceptors leads to the opening of TRPM1, in turn leading to ON bipolar cell depolarization, giving rise to the b-wave. Mutations in GRM6 lead to the loss of mGluR6 at the cell surface of the ON bipolar cells, resulting in the failure of depolarization of these cells and thus a severely reduced b-wave and complete CSNB [2]. Likewise, mutations in NYX, GPR179, and LRIT3, may cause complete CSNB [1].…”

“…CSNB is clinically and genetically heterogeneous. Some forms are associated with poor visual acuity, myopia, nystagmus, strabismus and fundus abnormalities [2][3][4]. CSNB is referred to as "complete" (MIM#613216), when there is no full-field electroretinography (full-field ERG) rod response combined with an electronegative rod-cone ERG with a normal awave (emanating from the photoreceptors) and severely reduced b -wave [1].…”

Interdomain movements in metabotropic glutamate receptor activation

“…Although absent in the GABA B receptor, several lines of evidence highlight the crucial role of the CRD in the allosteric connection between the VFT and the 7TM in the other class C GPCRs. First, genetic mutations of cysteines in mGlu6 and CaSR CRDs have been identified in human diseases, such as "night blindness" (19) and "hypocalciuric hypercalcemia" (20)(21)(22), respectively. Second, deletion of the CRD in mGluRs results in a loss of function (23).…”