Agosto, Melina
Permanent URI for this collectionhttps://hdl.handle.net/10222/85133
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Item Open Access The mGluR6 ligand-binding domain, but not the C-terminal domain, is required for synaptic localization in retinal ON-bipolar cells(2021) Agosto, MA; Adeosun, AAR; Kumar, N; Wensel, TGSignals from retinal photoreceptors are processed in two parallel channels-the ON channel responds to light increments, while the OFF channel responds to light decrements. The ON pathway is mediated by ON type bipolar cells (BCs), which receive glutamatergic synaptic input from photoreceptors via a G-protein-coupled receptor signaling cascade. The metabotropic glutamate receptor mGluR6 is located at the dendritic tips of all ON-BCs and is required for synaptic transmission. Thus, it is critically important for delivery of information from photoreceptors into the ON pathway. In addition to detecting glutamate, mGluR6 participates in interactions with other postsynaptic proteins, as well as trans-synaptic interactions with presynaptic ELFN proteins. Mechanisms of mGluR6 synaptic targeting and functional interaction with other synaptic proteins are unknown. Here, we show that multiple regions in the mGluR6 ligand-binding domain are necessary for both synaptic localization in BCs and ELFN1 binding in vitro. However, these regions were not required for plasma membrane localization in heterologous cells, indicating that secretory trafficking and synaptic localization are controlled by different mechanisms. In contrast, the mGluR6 C-terminus was dispensable for synaptic localization. In mGluR6 null mice, localization of the postsynaptic channel protein TRPM1 was compromised. Introducing WT mGluR6 rescued TRPM1 localization, while a C-terminal deletion mutant had significantly reduced rescue ability. We propose a model in which trans-synaptic ELFN1 binding is necessary for mGluR6 postsynaptic localization, whereas the C-terminus has a role in mediating TRPM1 trafficking. These findings reveal different sequence determinants of the multifunctional roles of mGluR6 in ON-BCs.Item Open Access Defective glycosylation and ELFN1 binding of mGluR6 congenital stationary night blindness mutants(2024) Pindwarawala M; Abid FA; Lee J; Miller ML; Noppers JS; Rideout AP; Agosto MASynaptic transmission from photoreceptors to ON-bipolar cells (BCs) requires the postsynaptic metabotropic glutamate receptor mGluR6, located at BC dendritic tips. Binding of the neurotransmitter glutamate initiates G protein signaling that regulates the TRPM1 transduction channel. mGluR6 also interacts with presynaptic ELFN adhesion proteins, and these interactions are important for mGluR6 synaptic localization. The mechanisms of mGluR6 trafficking and synaptic targeting remain poorly understood. In this study, we investigated mGluR6 missense mutations from patients with congenital stationary night blindness (CSNB), which is associated with loss of synaptic transmission to ON-BCs. We found that multiple CSNB mutations in the extracellular ligand-binding domain of mGluR6 impart a trafficking defect leading to lack of complex N-glycosylation but efficient plasma membrane insertion, suggesting a Golgi bypass mechanism. These mutants fail to bind ELFN1, consistent with lack of a necessary modification normally acquired in the Golgi. The same mutants were mislocalized in bipolar cells, explaining the loss of function in CSNB. The results reveal a key role of Golgi trafficking in mGluR6 function, and suggest a role of the extracellular domain in Golgi sorting.Item Open Access Complex N-glycosylation of mGluR6 is required for trans-synaptic interaction with ELFN adhesion proteins(2024) Miller ML; Pindwarawala M; Agosto MASynaptic transmission from retinal photoreceptors to downstream ON-type bipolar cells (BCs) depends on the postsynaptic metabotropic glutamate receptor mGluR6, located at the BC dendritic tips. Glutamate binding to mGluR6 initiates G-protein signaling that ultimately leads to BC depolarization in response to light. The mGluR6 receptor also engages in trans-synaptic interactions with presynaptic ELFN adhesion proteins. The roles of post-translational modifications in mGluR6 trafficking and function are unknown. Treatment with glycosidase enzymes PNGase F and Endo H demonstrated that both endogenous and heterologously expressed mGluR6 contain complex N-glycosylation acquired in the Golgi. Pull-down experiments with ELFN1 and ELFN2 extracellular domains revealed that these proteins interact exclusively with the complex glycosylated form of mGluR6. Mutation of the four predicted N-glycosylation sites, either singly or in combination, revealed that all four sites are glycosylated. Single mutations partially reduced, but did not abolish, surface expression in heterologous cells, while triple mutants had little or no surface expression, indicating that no single glycosylation site is necessary or sufficient for plasma membrane trafficking. Mutation at N445 severely impaired both ELFN1 and ELFN2 binding. All single mutants exhibited dendritic tip enrichment in rod BCs, as did the triple mutant with N445 as the sole N-glycosylation site, demonstrating that glycosylation at N445 is sufficient but not necessary for dendritic tip localization. The quadruple mutant was completely mislocalized. These results reveal a key role for complex N-glycosylation in regulating mGluR6 trafficking and ELFN binding, and by extension, function of the photoreceptor synapses.