| Popis: |
The metabotropic glutamate receptor 6 (mGluR6) is a G protein coupled receptor with expression restricted to retinal ON bipolar cells. mGluR6, a member of the Group III mGluRs, serves as the primary postsynaptic neurotransmitter receptor in these cells, which receive glutamatergic input directly from photoreceptor cells. Activated mGluR6 initiates a signaling cascade through inhibitory G proteins that results in inhibition of a cation channel and hyperpolarization of ON bipolar cells. Defects in mGluR6 expression result in congenital night blindness.Calcium current modulation mediated by activated mGluR6 can be measured using whole cell patch-clamp electrophysiology and a reconstitution system in sympathetic neurons from the rat superior cervical ganglion. Using this system, I examined coupling of mGluR6 with individual inhibitory G-alpha family members which were made resistant to pertussis toxin while inactivating the endogenous inhibitory G-proteins with pertussis toxin. The expression profile of each G-alpha in ON bipolar cells was also examined using single cell RT-PCR. These data strongly suggest that physiological signaling through mGluR6 in ON bipolar cells occurs predominantly via Go-alpha. To identify potential regulators of mGluR6 signaling, yeast two-hybrid screens were developed using the intracellular loops and C-terminus of mGluR6 as baits against a rat retinal cDNA library. CIP98 (CASK interacting protein, ‘whirlin’) was identified multiple times from these screens and showed specificity to Group III mGluRs. CIP98 is a largely uncharacterized scaffolding protein containing 3 PDZ domains (domains known to interact with G protein coupled receptors) and a proline-rich domain. Expression of CIP98 was verified in ON bipolar cells using single cell RT-PCR and in whole retina extracts using immunoblotting. Using our previously established neuronal reconstitution system we found a dose-dependent shift in the mGluR6 response to L-glutamate when co-expressed with truncated CIP98 isoforms containing intact PDZ domains. To confirm and further characterize CIP98 - mGluR6 interaction, co-immunoprecipitation and immunocytochemistry experiments were performed using HEK293 cells. Though the co-immunoprecipitation experiments were inconclusive, significant co-localization between CIP98 and mGluR6 was detected by immunofluorescence. Together, my data suggest that CIP98 is a potential regulator of mGluR6 signaling. |
