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.
Wang
L
L
Hormone Research Center and Department of Biology, Chonnam National University, Kwangju, 500-757, Republic of Korea.
Oh
D Y
DY
Bogerd
J
J
Choi
H S
HS
Ahn
R S
RS
Seong
J Y
JY
Kwon
H B
HB
D017398:Alternative Splicing; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation / Q000502:physiology*; D006605:Hibernation / Q000502:physiology; D008024:Ligands; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D011892:Rana catesbeiana / Q000235:genetics / Q000502:physiology*; D011966:Receptors, LHRH / Q000235:genetics / Q000502:physiology*; D012016:Reference Values; D012098:Reproduction / Q000502:physiology; D012621:Seasons; D015398:Signal Transduction / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology
C434455:GnRH receptor 3; D008024:Ligands; D011966:Receptors, LHRH; D012313:RNA