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

Li Wang, Da Y. Oh, Jan Bogerd, Hueng S. Choi, Ryun S. Ahn, Jae Young Seong, Hyuk B. Kwon

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)4015-4025
Number of pages11
JournalEndocrinology
Volume142
Issue number9
DOIs
Publication statusPublished - 2001 Sep 12
Externally publishedYes

Fingerprint

Rana catesbeiana
LHRH Receptors
Protein Isoforms
Cytoplasm
Cell Membrane
Hibernation
Proteins
Alternative Splicing
Amphibians
Green Fluorescent Proteins
HeLa Cells
Gonadotropin-Releasing Hormone
Introns
Exons
Signal Transduction
Ligands
Messenger RNA
GnRH receptor 3

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling. / Wang, Li; Oh, Da Y.; Bogerd, Jan; Choi, Hueng S.; Ahn, Ryun S.; Seong, Jae Young; Kwon, Hyuk B.

In: Endocrinology, Vol. 142, No. 9, 12.09.2001, p. 4015-4025.

Research output: Contribution to journalArticle

Wang, Li ; Oh, Da Y. ; Bogerd, Jan ; Choi, Hueng S. ; Ahn, Ryun S. ; Seong, Jae Young ; Kwon, Hyuk B. / Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling. In: Endocrinology. 2001 ; Vol. 142, No. 9. pp. 4015-4025.
@article{603c2f571e8c4001bb56520f3a260a9e,
title = "Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling",
abstract = "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.",
author = "Li Wang and Oh, {Da Y.} and Jan Bogerd and Choi, {Hueng S.} and Ahn, {Ryun S.} and Seong, {Jae Young} and Kwon, {Hyuk B.}",
year = "2001",
month = "9",
day = "12",
doi = "10.1210/en.142.9.4015",
language = "English",
volume = "142",
pages = "4015--4025",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "9",

}

TY - JOUR

T1 - Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling

AU - Wang, Li

AU - Oh, Da Y.

AU - Bogerd, Jan

AU - Choi, Hueng S.

AU - Ahn, Ryun S.

AU - Seong, Jae Young

AU - Kwon, Hyuk B.

PY - 2001/9/12

Y1 - 2001/9/12

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034870177&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034870177&partnerID=8YFLogxK

U2 - 10.1210/en.142.9.4015

DO - 10.1210/en.142.9.4015

M3 - Article

C2 - 11517181

AN - SCOPUS:0034870177

VL - 142

SP - 4015

EP - 4025

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 9

ER -