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Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development.

Tallquist MD, French WJ, Soriano P - PLoS Biol. (2003)

Bottom Line: A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p.Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal.The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. michelle.tallquist@utsouthwestern.edu

ABSTRACT
The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.

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Biochemistry of MEFs from F7 and F1 Mice(A) Whole-cell lysates were generated from MEFs that were unstimulated or stimulated with PDGFAA and/or PDGFBB alone, 100 ng/ml and 30 ng/ml, respectively. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the anti-phosphotyrosine antibody (4G10).(B) Immunoprecipitation of tyrosine-phosphorylated proteins from wild-type and the F7 series of mutant MEFs. The precipitates were then run on SDS–PAGE, and a Western blot was performed using anti-Src [pY418] antibody and anti-PDGFRβ 97A.(C) Whole-cell lysates from unstimulated or stimulated MEFs. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the indicated phosphorylated-specific antibodies. Blots were stripped and blotted with antibodies to the corresponding unphosphorylated proteins to demonstrate protein loading. Data are representative of multiple experiments from at least two independently derived cell lines.(D) Western blots of whole-cell lysates from 5 ×104 cells per lane blotted with antibodies to PDGFRβ (06-498) and ERK as a loading control.
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pbio.0000052-g010: Biochemistry of MEFs from F7 and F1 Mice(A) Whole-cell lysates were generated from MEFs that were unstimulated or stimulated with PDGFAA and/or PDGFBB alone, 100 ng/ml and 30 ng/ml, respectively. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the anti-phosphotyrosine antibody (4G10).(B) Immunoprecipitation of tyrosine-phosphorylated proteins from wild-type and the F7 series of mutant MEFs. The precipitates were then run on SDS–PAGE, and a Western blot was performed using anti-Src [pY418] antibody and anti-PDGFRβ 97A.(C) Whole-cell lysates from unstimulated or stimulated MEFs. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the indicated phosphorylated-specific antibodies. Blots were stripped and blotted with antibodies to the corresponding unphosphorylated proteins to demonstrate protein loading. Data are representative of multiple experiments from at least two independently derived cell lines.(D) Western blots of whole-cell lysates from 5 ×104 cells per lane blotted with antibodies to PDGFRβ (06-498) and ERK as a loading control.

Mentions: Because F2, F3, and F5 mutant receptors have been previously studied biochemically (Valius and Kazlauskas 1993; Heuchel et al. 1999; Tallquist et al. 2000), we have focused our biochemical analysis on the F1 and F7 mutant receptors' signal transduction to verify the effects of these particular mutations on downstream signal transduction cascades. We have used mouse embryo fibroblasts (MEFs) for these analyses. All lines of MEFs that we generated expressed the PDGFRβ at similar levels (Figure 10D) as well as the PDGFRα (data not shown). To avoid stimulation of the PDGFRα by PDGFBB, we downregulated PDGFRα surface expression by pretreatment with PDGFAA 2 h before PDGFBB stimulation. In all cell lines examined, we observed an increase in tyrosine phosphorylation in response to ligand (Figure 10A). The most evident phosphorylated bands are around 200 kd, which are likely to be the PDGFRα and PDGFRβ. Although we have mutated seven of the 13 tyrosines, a significant amount of phosphorylation is observed in all cell lines, albeit at lower levels in the F7/− cell line (Figure 10A and 10B). In the whole-cell lysate phosphotyrosine blot, the phosphorylated protein detected at 200 kd is likely cytoplasmic PDGFRα, as it is reduced in F7 cells after downregulation of the PDGFRα.


Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development.

Tallquist MD, French WJ, Soriano P - PLoS Biol. (2003)

Biochemistry of MEFs from F7 and F1 Mice(A) Whole-cell lysates were generated from MEFs that were unstimulated or stimulated with PDGFAA and/or PDGFBB alone, 100 ng/ml and 30 ng/ml, respectively. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the anti-phosphotyrosine antibody (4G10).(B) Immunoprecipitation of tyrosine-phosphorylated proteins from wild-type and the F7 series of mutant MEFs. The precipitates were then run on SDS–PAGE, and a Western blot was performed using anti-Src [pY418] antibody and anti-PDGFRβ 97A.(C) Whole-cell lysates from unstimulated or stimulated MEFs. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the indicated phosphorylated-specific antibodies. Blots were stripped and blotted with antibodies to the corresponding unphosphorylated proteins to demonstrate protein loading. Data are representative of multiple experiments from at least two independently derived cell lines.(D) Western blots of whole-cell lysates from 5 ×104 cells per lane blotted with antibodies to PDGFRβ (06-498) and ERK as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261889&req=5

pbio.0000052-g010: Biochemistry of MEFs from F7 and F1 Mice(A) Whole-cell lysates were generated from MEFs that were unstimulated or stimulated with PDGFAA and/or PDGFBB alone, 100 ng/ml and 30 ng/ml, respectively. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the anti-phosphotyrosine antibody (4G10).(B) Immunoprecipitation of tyrosine-phosphorylated proteins from wild-type and the F7 series of mutant MEFs. The precipitates were then run on SDS–PAGE, and a Western blot was performed using anti-Src [pY418] antibody and anti-PDGFRβ 97A.(C) Whole-cell lysates from unstimulated or stimulated MEFs. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the indicated phosphorylated-specific antibodies. Blots were stripped and blotted with antibodies to the corresponding unphosphorylated proteins to demonstrate protein loading. Data are representative of multiple experiments from at least two independently derived cell lines.(D) Western blots of whole-cell lysates from 5 ×104 cells per lane blotted with antibodies to PDGFRβ (06-498) and ERK as a loading control.
Mentions: Because F2, F3, and F5 mutant receptors have been previously studied biochemically (Valius and Kazlauskas 1993; Heuchel et al. 1999; Tallquist et al. 2000), we have focused our biochemical analysis on the F1 and F7 mutant receptors' signal transduction to verify the effects of these particular mutations on downstream signal transduction cascades. We have used mouse embryo fibroblasts (MEFs) for these analyses. All lines of MEFs that we generated expressed the PDGFRβ at similar levels (Figure 10D) as well as the PDGFRα (data not shown). To avoid stimulation of the PDGFRα by PDGFBB, we downregulated PDGFRα surface expression by pretreatment with PDGFAA 2 h before PDGFBB stimulation. In all cell lines examined, we observed an increase in tyrosine phosphorylation in response to ligand (Figure 10A). The most evident phosphorylated bands are around 200 kd, which are likely to be the PDGFRα and PDGFRβ. Although we have mutated seven of the 13 tyrosines, a significant amount of phosphorylation is observed in all cell lines, albeit at lower levels in the F7/− cell line (Figure 10A and 10B). In the whole-cell lysate phosphotyrosine blot, the phosphorylated protein detected at 200 kd is likely cytoplasmic PDGFRα, as it is reduced in F7 cells after downregulation of the PDGFRα.

Bottom Line: A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p.Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal.The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. michelle.tallquist@utsouthwestern.edu

ABSTRACT
The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.

Show MeSH