A Preliminary Study On The Function Of Vlgr1 In Auditory Systems

G protein-coupled receptors (GPCR) are seven trans-membrane helical proteins, which sense the extracellular stimuli and regulate cellular responses. The genomic projects have identified GPCRs as one of the largest protein super-families. GPCRs play important roles in visual, olfactory and gustatory systems; in fact, visual, olfactory and some gustatory receptors are GPCRs. But the role of GPCRs in auditory system is less understood.In this research, detection of the expression of GPCR mRNAs in cochlear tissue of murine through RT-PCR shows that several GPCR mRNAs are widely expressed in the cochlear tissue of murine, including Vlgr1 (Very large G protein-coupled receptor 1). Vlgr1 is the largest GPCR identified to date; its mRNA spans 19kb and encodes 6300 amino acids. Vlgr1 is a core component of ankle-link complex in inner ear hair cells. Human genetics data suggested that Vlgr1 mutations cause Usher syndromes and seizures. Although significant progresses have been made, the details of Vlgr1’s function in hair cells, its signaling cascade, and the mechanisms underlying causative effects of Vlgr1 mutations in human diseases remain elusive and ask for further investigation.To further investigate the function of Vlgr1 in the auditory system, first we use Two-Hybrid System to find the protein interact to the Vlgr1 in the inner ear, the results showed that Vlgr1 autoactivates the reporter genes, so could not be used in yeast two-hybrid screening.Vlgr1 is an EAR domain-containing protein. Besides Vlgr1, Tspear and LGI1-4 are also EAR domain-containing protein. EAR domain may play an important role in mediating these protein interact with each other. We use Co-immunoprecipitation assay to verify the interaction, the result shows that the EAR domain of Vlgr1 have strong homodimerization. It exist in the homodimer form after cell over-expression assay.In order to further investigate the Vlgr1 animal level physiological functions in the auditory system, we introduce Vlgr1/del7TM mice from The Jackson Laboratory. Currently we have identifiable the genotype of the mice, then we will use the Microarray and protein profiling assay to analyze mRNAs and protein expression in tissue of the Vlgrl/del7TM mice inner ear, then we could possible find the downstream gene regulated by Vlgrl.