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Inhibitory Activity of Alternative Splice Variants of the Bullfrog GnRH Receptor-3 on Wild-Type Receptor Signaling

Hormone Research Center and Department of Biology (L.W., D.Y.O., H.S.C., R.S.A., J.Y.S., H.B.K.), Chonnam National University, Kwangju, 500–757, Republic of Korea; and the Research Group for Comparative Endocrinology (J.B.), Department of Experimental Zoology, Utrecht University, Utrecht 3584, The Netherlands

Address all correspondence and requests for reprints to: Hyuk Bang Kwon, Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea. E-mail: kwonhb{at}chonnam.chonnam.ac.kr

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.

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