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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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Targeting Strategy and Southern Blot(A) Targeting vector used to create F5 mutant allele. Two exons contain all five mutated tyrosines.(B) Targeting vector containing mutations in 5′ exons used to generate the F7 mutant allele.(C) Wild-type allele.(D) Targeted allele with PGK–neo removal. Restriction enzyme abbreviations: Sp, SpeI; A, Asp718; S, SacI; RV, EcoRV; H, HindIII; X, XhoI; and RI, EcoRI. Green boxes indicate probes used in Southern blotting for F7-targeted ES cells. The blue arrow indicates the exon where point mutation causes frameshift in truncation mutation. Black boxes indicate wild-type exons. Red boxes indicate exons containing targeted mutations. Restriction enzymes in red indicate sites introduced by mutagenesis to verify proper homologous recombination by Southern blotting. Circles denote FRT sites. Triangles denote loxP sites.(E) Southern blot results from various ES cell lines. SpeI digest using P1 probe. Blot with P2 probe gave expected results (data not shown). Lanes 1 and 3, F5 allele targeted. Lanes 2 and 4, wild-type allele targeted. Lanes 5 and 7, F5 mutant ES cells after and before Cre activity, respectively. Lane 6, wild-type ES cell clone.
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pbio.0000052-g002: Targeting Strategy and Southern Blot(A) Targeting vector used to create F5 mutant allele. Two exons contain all five mutated tyrosines.(B) Targeting vector containing mutations in 5′ exons used to generate the F7 mutant allele.(C) Wild-type allele.(D) Targeted allele with PGK–neo removal. Restriction enzyme abbreviations: Sp, SpeI; A, Asp718; S, SacI; RV, EcoRV; H, HindIII; X, XhoI; and RI, EcoRI. Green boxes indicate probes used in Southern blotting for F7-targeted ES cells. The blue arrow indicates the exon where point mutation causes frameshift in truncation mutation. Black boxes indicate wild-type exons. Red boxes indicate exons containing targeted mutations. Restriction enzymes in red indicate sites introduced by mutagenesis to verify proper homologous recombination by Southern blotting. Circles denote FRT sites. Triangles denote loxP sites.(E) Southern blot results from various ES cell lines. SpeI digest using P1 probe. Blot with P2 probe gave expected results (data not shown). Lanes 1 and 3, F5 allele targeted. Lanes 2 and 4, wild-type allele targeted. Lanes 5 and 7, F5 mutant ES cells after and before Cre activity, respectively. Lane 6, wild-type ES cell clone.

Mentions: Previous studies of the PDGFRβ have revealed an essential role for this receptor in v/p development, but attempts to identify essential biochemical signals thus far have demonstrated that loss of certain signaling pathways only diminishes PDGFRβ-driven responses (Heuchel et al. 1999; Tallquist et al. 2000). To study key signaling pathways, we have generated an allelic series of PDGFRβ mutants. Figure 1 illustrates the mutations that we have generated in the PDGFRβ locus and the signaling pathways that are disrupted by these mutations. Each mutant will be referred to by the number of tyrosines (Y) that have been mutated. For example, the mutation in the RasGAP-binding site is the PDGFRβF1/F1 or F1/F1 mutant. The truncation mutation of the PDGFRβ (βT) was created by the introduction of a frameshift and subsequent premature stop codon downstream of the RasGAP-binding site. Figure 2 illustrates the targeting events that were used to generate the series of mutants. The F1-, F2-, F3-, F5-, and βT-targeted mutations were generated by engineering Y–F, Y–I, or frameshift mutations in the same targeting vector (Figure 2A). The F7 mutation was generated by targeting the F5 heterozygous embryonic stem (ES) cells (Figure 2B; see Materials and Methods). Cells that contained all mutations on the same allele, as determined by Southern blotting, were used to generate the F7 line. All mutant mice were viable and fertile as homozygotes except the truncation allele, βT, which lacks the second half of the kinase domain and the SHP-2- and PLCγ-binding sites. Based on a similar mutation in the PDGFRβ, we assume that this receptor is kinase deficient and incapable of inducing DNA synthesis, but it still should bind ligand and undergo receptor downregulation (Escobedo et al. 1988). Embryos homozygous for the βT allele die perinatally with a phenotype identical to that of the PDGFRβ embryos. E18 embryos exhibit edema and hemorrhaging in multiple tissues, including the kidney, brain, and skin (data not shown). These results suggest that PDGFRβ kinase activity is required for v/p development and that the receptor cannot function in the absence of kinase activity, unlike another RTK, vascular endothelial cell growth factor receptor 1 (Hiratsuka et al. 1998).


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

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

Targeting Strategy and Southern Blot(A) Targeting vector used to create F5 mutant allele. Two exons contain all five mutated tyrosines.(B) Targeting vector containing mutations in 5′ exons used to generate the F7 mutant allele.(C) Wild-type allele.(D) Targeted allele with PGK–neo removal. Restriction enzyme abbreviations: Sp, SpeI; A, Asp718; S, SacI; RV, EcoRV; H, HindIII; X, XhoI; and RI, EcoRI. Green boxes indicate probes used in Southern blotting for F7-targeted ES cells. The blue arrow indicates the exon where point mutation causes frameshift in truncation mutation. Black boxes indicate wild-type exons. Red boxes indicate exons containing targeted mutations. Restriction enzymes in red indicate sites introduced by mutagenesis to verify proper homologous recombination by Southern blotting. Circles denote FRT sites. Triangles denote loxP sites.(E) Southern blot results from various ES cell lines. SpeI digest using P1 probe. Blot with P2 probe gave expected results (data not shown). Lanes 1 and 3, F5 allele targeted. Lanes 2 and 4, wild-type allele targeted. Lanes 5 and 7, F5 mutant ES cells after and before Cre activity, respectively. Lane 6, wild-type ES cell clone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261889&req=5

pbio.0000052-g002: Targeting Strategy and Southern Blot(A) Targeting vector used to create F5 mutant allele. Two exons contain all five mutated tyrosines.(B) Targeting vector containing mutations in 5′ exons used to generate the F7 mutant allele.(C) Wild-type allele.(D) Targeted allele with PGK–neo removal. Restriction enzyme abbreviations: Sp, SpeI; A, Asp718; S, SacI; RV, EcoRV; H, HindIII; X, XhoI; and RI, EcoRI. Green boxes indicate probes used in Southern blotting for F7-targeted ES cells. The blue arrow indicates the exon where point mutation causes frameshift in truncation mutation. Black boxes indicate wild-type exons. Red boxes indicate exons containing targeted mutations. Restriction enzymes in red indicate sites introduced by mutagenesis to verify proper homologous recombination by Southern blotting. Circles denote FRT sites. Triangles denote loxP sites.(E) Southern blot results from various ES cell lines. SpeI digest using P1 probe. Blot with P2 probe gave expected results (data not shown). Lanes 1 and 3, F5 allele targeted. Lanes 2 and 4, wild-type allele targeted. Lanes 5 and 7, F5 mutant ES cells after and before Cre activity, respectively. Lane 6, wild-type ES cell clone.
Mentions: Previous studies of the PDGFRβ have revealed an essential role for this receptor in v/p development, but attempts to identify essential biochemical signals thus far have demonstrated that loss of certain signaling pathways only diminishes PDGFRβ-driven responses (Heuchel et al. 1999; Tallquist et al. 2000). To study key signaling pathways, we have generated an allelic series of PDGFRβ mutants. Figure 1 illustrates the mutations that we have generated in the PDGFRβ locus and the signaling pathways that are disrupted by these mutations. Each mutant will be referred to by the number of tyrosines (Y) that have been mutated. For example, the mutation in the RasGAP-binding site is the PDGFRβF1/F1 or F1/F1 mutant. The truncation mutation of the PDGFRβ (βT) was created by the introduction of a frameshift and subsequent premature stop codon downstream of the RasGAP-binding site. Figure 2 illustrates the targeting events that were used to generate the series of mutants. The F1-, F2-, F3-, F5-, and βT-targeted mutations were generated by engineering Y–F, Y–I, or frameshift mutations in the same targeting vector (Figure 2A). The F7 mutation was generated by targeting the F5 heterozygous embryonic stem (ES) cells (Figure 2B; see Materials and Methods). Cells that contained all mutations on the same allele, as determined by Southern blotting, were used to generate the F7 line. All mutant mice were viable and fertile as homozygotes except the truncation allele, βT, which lacks the second half of the kinase domain and the SHP-2- and PLCγ-binding sites. Based on a similar mutation in the PDGFRβ, we assume that this receptor is kinase deficient and incapable of inducing DNA synthesis, but it still should bind ligand and undergo receptor downregulation (Escobedo et al. 1988). Embryos homozygous for the βT allele die perinatally with a phenotype identical to that of the PDGFRβ embryos. E18 embryos exhibit edema and hemorrhaging in multiple tissues, including the kidney, brain, and skin (data not shown). These results suggest that PDGFRβ kinase activity is required for v/p development and that the receptor cannot function in the absence of kinase activity, unlike another RTK, vascular endothelial cell growth factor receptor 1 (Hiratsuka et al. 1998).

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