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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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Importance of the spacing of Glu residues. (A) A series  of clones, based on the mutant CONS.C2xE, were constructed  varying the spacing between two Glu residues, from a minimum  spacing of two residues (diad mutant) to a maximum spacing of  eight residues (octad mutant). (B) Transformation by each isolate shown in (A) was quantitated as a percentage of p185neu. Numerical results of transformation assays are presented graphically  and represent the average values from two independent experiments, normalized by cotransfection with pSV2neo.
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Figure 5: Importance of the spacing of Glu residues. (A) A series of clones, based on the mutant CONS.C2xE, were constructed varying the spacing between two Glu residues, from a minimum spacing of two residues (diad mutant) to a maximum spacing of eight residues (octad mutant). (B) Transformation by each isolate shown in (A) was quantitated as a percentage of p185neu. Numerical results of transformation assays are presented graphically and represent the average values from two independent experiments, normalized by cotransfection with pSV2neo.

Mentions: The design of the degenerate pools of transmembrane domains, as well as the design of the subsequent consensus repeat clones described above, assumed without any real evidence that there would exist a fundamental heptad structural motif resulting from the α-helical transmembrane domain. To directly examine this premise, we constructed a series of clones in which we varied the spacing between Glu residues. As the parental clone for this series, we chose a clone with two Glu residues spaced seven residues apart in a transmembrane domain composed otherwise of only Val residues, designated CONS.C2xE. The spacing between the two Glu residues was then varied from two to eight residues, as shown in Fig. 5 A. The results of transformation assays with these clones revealed a dramatic effect of spacing, with the heptad spacing yielding significantly greater transforming activity than any of the other clones (Fig. 5 B). When cells expressing these constructs were examined by indirect immunofluorescence, all constructs exhibited cell surface expression (data not shown), indicating that the lack of transformation was not due to a defect in surface localization. This experiment provides compelling evidence for a basic heptad structural motif in the transmembrane domain of activated forms of p185c-neu.


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)

Importance of the spacing of Glu residues. (A) A series  of clones, based on the mutant CONS.C2xE, were constructed  varying the spacing between two Glu residues, from a minimum  spacing of two residues (diad mutant) to a maximum spacing of  eight residues (octad mutant). (B) Transformation by each isolate shown in (A) was quantitated as a percentage of p185neu. Numerical results of transformation assays are presented graphically  and represent the average values from two independent experiments, normalized by cotransfection with pSV2neo.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Importance of the spacing of Glu residues. (A) A series of clones, based on the mutant CONS.C2xE, were constructed varying the spacing between two Glu residues, from a minimum spacing of two residues (diad mutant) to a maximum spacing of eight residues (octad mutant). (B) Transformation by each isolate shown in (A) was quantitated as a percentage of p185neu. Numerical results of transformation assays are presented graphically and represent the average values from two independent experiments, normalized by cotransfection with pSV2neo.
Mentions: The design of the degenerate pools of transmembrane domains, as well as the design of the subsequent consensus repeat clones described above, assumed without any real evidence that there would exist a fundamental heptad structural motif resulting from the α-helical transmembrane domain. To directly examine this premise, we constructed a series of clones in which we varied the spacing between Glu residues. As the parental clone for this series, we chose a clone with two Glu residues spaced seven residues apart in a transmembrane domain composed otherwise of only Val residues, designated CONS.C2xE. The spacing between the two Glu residues was then varied from two to eight residues, as shown in Fig. 5 A. The results of transformation assays with these clones revealed a dramatic effect of spacing, with the heptad spacing yielding significantly greater transforming activity than any of the other clones (Fig. 5 B). When cells expressing these constructs were examined by indirect immunofluorescence, all constructs exhibited cell surface expression (data not shown), indicating that the lack of transformation was not due to a defect in surface localization. This experiment provides compelling evidence for a basic heptad structural motif in the transmembrane domain of activated forms of p185c-neu.

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