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Isolation and characterization of a transforming growth factor-beta Type II receptor cDNA from Xenopus laevis
S M Dhanasekaran1, U D Vempati, P Kondaiah
1Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560 012, India.
Abstract:
Transforming Growth Factor-beta (TGF-beta) and their receptors have been characterized from many organisms. Two TGF-beta signaling receptors called Type I and II have been described for various ligands of the superfamily from organisms ranging from Drosophila to humans. In Xenopus laevis, TGF-beta2 and 5 have been reported and presumably, play important roles during early development. Several Type I and type II receptors for many ligands of the TGF-beta superfamily except TGF-beta type II receptor (TbetaIIR), have been characterized in Xenopus laevis. A chemical cross linking experiment using iodinated TGF-beta1 and -beta5, revealed four specific binding proteins on XTC cells. In order to understand the TGF-beta involvement during Xenopus development, a TGF-beta type II receptor (XTbetaIIR) has been isolated from a XTC cDNA library. XTbetaIIR was a partial cDNA lacking a portion of the signal peptide. The sequence analysis and homology comparison with the human TbetaIIR revealed 67% amino acid similarity in the extra cellular domain, 60% similarity in the transmembrane domain and 87% similarity in the cytoplasmic kinase domain, suggesting that XTbetaIIR is a putative TGF-beta type II receptor. In addition, the consensus amino acid motif for serine threonine receptor kinases was also present. Further, a dominant negative expression construct lacking the cytoplasmic kinase domain (engineered with the signal peptide from human TGF-beta type II receptor), was able to abolish TGF-beta mediated induction of a luciferase reporter plasmid, in a transient cell transfection assay. This substantiates the notion that XTbetaIIR cDNA can act as a receptor for TGF-beta. RT-PCR analysis using RNA isolated from various developmental stages of Xenopus laevis revealed expression of this gene in all the early stages of development and in the adult organs, except in stages 46/48.
Insights
Researchers identified a novel Transforming Growth Factor-beta type II receptor (XTbetaIIR) in Xenopus laevis. This receptor is crucial for early development and demonstrates high similarity to human counterparts.
Area of Science:
- Developmental Biology
- Molecular Biology
- Cell Signaling
Background:
- Transforming Growth Factor-beta (TGF-beta) signaling is vital in development.
- Type I and II receptors mediate TGF-beta superfamily signaling.
- While many TGF-beta receptors are known, the type II receptor in Xenopus laevis was previously uncharacterized.
Purpose of the Study:
- To isolate and characterize the TGF-beta type II receptor (XTbetaIIR) in Xenopus laevis.
- To investigate the role of XTbetaIIR in early Xenopus development.
Main Methods:
- Isolation of XTbetaIIR cDNA from a Xenopus laevis cell line (XTC) library.
- Sequence analysis and homology comparison with human TbetaIIR.
- Functional analysis using a dominant-negative expression construct and luciferase reporter assay.
- Gene expression analysis via RT-PCR.
Main Results:
- A partial XTbetaIIR cDNA was isolated, showing significant amino acid similarity to human TbetaIIR (up to 87% in the cytoplasmic kinase domain).
- The XTbetaIIR sequence contains a conserved serine/threonine kinase motif.
- A dominant-negative construct confirmed XTbetaIIR's role as a functional TGF-beta receptor.
- XTbetaIIR gene expression was detected in all early developmental stages and adult organs, except for late stages (46/48).
Conclusions:
- XTbetaIIR is a functional TGF-beta type II receptor in Xenopus laevis.
- This receptor plays a significant role throughout Xenopus development.
- The findings provide insights into TGF-beta signaling pathways in amphibian development.