Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling

Recently we characterized three distinct GnRH receptors in the bullfrog (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we further investigated the expression and function of splice variants, generated from the primary bfGnRHR-3 transcript by exon skipping (splice variant 1), intron retention (splice variants 2 and 3), and/or transcriptional slippage (splice variant 4), apart from the constitutively spliced form (wild-type). Cellular expression and function of the splice variants were examined using a transient expression system. Immunoblot analysis revealed that the wild-type receptor and all splice variant proteins were expressed in transfected HeLa cells with no significant differences in expression levels. These splice variants showed a very low binding affinity to ligand and did not induce signal transduction in response to GnRH treatment. Interestingly, cotransfection of the wild-type with splice variants 2-4, but not with splice variant 1, significantly inhibited wild-type receptor-mediated signaling. Subcellular localization analysis of green fluorescent protein-tagged wild-type and splice variant proteins revealed that the wild-type receptor protein was mainly localized in the cell membrane, whereas the splice variant 1 protein was exclusively detected in the cytoplasm. The splice variant 2-4 proteins, however, were found in both the cell membrane and cytoplasm. The inhibition of wild-type receptor signaling by splice variants 2-4 and the subcellular localization of splice variants 2-4 suggest a possible physical interaction of splice variants 2-4 with the wild-type receptor protein. In addition, the ratio of mRNA levels of the wild-type to splice variants 2-4 significantly varied from hibernation (wild-type < splice variants 2-4) to the prebreeding season (wild-type > splice variants 2-4). Collectively, these results suggest that alternative splicing of the bfGnRHR-3 primary transcript plays a role in fine-tuning GnRH receptor function in amphibians.