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Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

Chen LI, Webster MK, Meyer AN, Donoghue DJ - J. Cell Biol. (1997)

Bottom Line: The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu.Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation.These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0367, USA.

ABSTRACT
The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

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Immunofluorescence of nontransforming isolates  DEG.6–DEG.10. Double-label indirect immunofluorescence was  used to detect either cell surface expression of p185c-neu-related  proteins (right) or, after permeabilization of cells, intracellular  expression (left). Conditions for the double-label immunofluorescence are described in Materials and Methods. (A) (A and B)  mutant DEG.6; (C and D) mutant DEG.7; (E and F) mutant  DEG.8; (G and H) mutant DEG.9; (I and J) mutant DEG.10. (B)  (A and B) Mock transfected cells; (C and D) p185neu.
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Figure 2: Immunofluorescence of nontransforming isolates DEG.6–DEG.10. Double-label indirect immunofluorescence was used to detect either cell surface expression of p185c-neu-related proteins (right) or, after permeabilization of cells, intracellular expression (left). Conditions for the double-label immunofluorescence are described in Materials and Methods. (A) (A and B) mutant DEG.6; (C and D) mutant DEG.7; (E and F) mutant DEG.8; (G and H) mutant DEG.9; (I and J) mutant DEG.10. (B) (A and B) Mock transfected cells; (C and D) p185neu.

Mentions: Approximately 15 nontransforming mutants were also isolated from Pool 1, five of which, DEG.6 through DEG.10, are described in Fig. 1 D. The distinctions between the transforming and nontransforming isolates were not entirely obvious. Double-label indirect immunofluorescence was used to determine whether the lack of transforming activity of these mutants might reflect an abnormal subcellular localization. This was accomplished by using an antibody against an extracellular epitope to detect surface expression in cells before permeabilization, followed by a second antibody against the COOH terminus to detect intracellular expression in the same cell after permeabilization (Fig. 2). Fig. 2 B shows the immunofluorescence observed for control cells under identical conditions, with mock-transfected cells negative for both intracellular and surface staining (Fig. 2 B, A and B), whereas cells expressing p185neu exhibit readily detectable staining both intracellularly and also at the cell surface (Fig. 2 B, C and D). As shown in Fig. 2 A, the mutants DEG.7, DEG.9, and DEG.10 were expressed intracellularly but failed to reach the cell surface (Fig. 2 A, D, H, and J), suggesting that their lack of biological activity might be due to aggregation or interaction with some component of the ER/Golgi. In contrast, the mutants DEG.6 and DEG.8 were expressed at the cell surface (Fig. 2 A, B and F), suggesting that their transmembrane domains fail to activate these receptors, despite localizing normally to the plasma membrane.


Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

Chen LI, Webster MK, Meyer AN, Donoghue DJ - J. Cell Biol. (1997)

Immunofluorescence of nontransforming isolates  DEG.6–DEG.10. Double-label indirect immunofluorescence was  used to detect either cell surface expression of p185c-neu-related  proteins (right) or, after permeabilization of cells, intracellular  expression (left). Conditions for the double-label immunofluorescence are described in Materials and Methods. (A) (A and B)  mutant DEG.6; (C and D) mutant DEG.7; (E and F) mutant  DEG.8; (G and H) mutant DEG.9; (I and J) mutant DEG.10. (B)  (A and B) Mock transfected cells; (C and D) p185neu.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139875&req=5

Figure 2: Immunofluorescence of nontransforming isolates DEG.6–DEG.10. Double-label indirect immunofluorescence was used to detect either cell surface expression of p185c-neu-related proteins (right) or, after permeabilization of cells, intracellular expression (left). Conditions for the double-label immunofluorescence are described in Materials and Methods. (A) (A and B) mutant DEG.6; (C and D) mutant DEG.7; (E and F) mutant DEG.8; (G and H) mutant DEG.9; (I and J) mutant DEG.10. (B) (A and B) Mock transfected cells; (C and D) p185neu.
Mentions: Approximately 15 nontransforming mutants were also isolated from Pool 1, five of which, DEG.6 through DEG.10, are described in Fig. 1 D. The distinctions between the transforming and nontransforming isolates were not entirely obvious. Double-label indirect immunofluorescence was used to determine whether the lack of transforming activity of these mutants might reflect an abnormal subcellular localization. This was accomplished by using an antibody against an extracellular epitope to detect surface expression in cells before permeabilization, followed by a second antibody against the COOH terminus to detect intracellular expression in the same cell after permeabilization (Fig. 2). Fig. 2 B shows the immunofluorescence observed for control cells under identical conditions, with mock-transfected cells negative for both intracellular and surface staining (Fig. 2 B, A and B), whereas cells expressing p185neu exhibit readily detectable staining both intracellularly and also at the cell surface (Fig. 2 B, C and D). As shown in Fig. 2 A, the mutants DEG.7, DEG.9, and DEG.10 were expressed intracellularly but failed to reach the cell surface (Fig. 2 A, D, H, and J), suggesting that their lack of biological activity might be due to aggregation or interaction with some component of the ER/Golgi. In contrast, the mutants DEG.6 and DEG.8 were expressed at the cell surface (Fig. 2 A, B and F), suggesting that their transmembrane domains fail to activate these receptors, despite localizing normally to the plasma membrane.

Bottom Line: The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu.Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation.These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0367, USA.

ABSTRACT
The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

Show MeSH
Related in: MedlinePlus