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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 number of Glu  residues. (A) Derivatives of CONS.A. Three  derivatives were constructed, each having a  single Glu residue. Depending upon the position of the Glu residue, these mutants were  designated CONS.AE2, CONS.AE3, and  CONS.AE4. (B) Derivatives of CONS.C. Derivatives were constructed in the background  of CONS.C having from one to four Glu residues. These clones are designated CONS.C1xE,  CONS.C2xE, CONS.C3xE, and CONS.C4xE.  Note that CONS.C3xE is the parental  CONS.C mutant presented in Fig. 3. Transformation by each isolate was quantitated as  a percentage of p185neu. Results represent the  average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as −, +, or ++ as described in Fig. 1. For transformation results  shown in B, numerical values are also shown in parentheses as the percentage of p185neu.
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Figure 6: Importance of the number of Glu residues. (A) Derivatives of CONS.A. Three derivatives were constructed, each having a single Glu residue. Depending upon the position of the Glu residue, these mutants were designated CONS.AE2, CONS.AE3, and CONS.AE4. (B) Derivatives of CONS.C. Derivatives were constructed in the background of CONS.C having from one to four Glu residues. These clones are designated CONS.C1xE, CONS.C2xE, CONS.C3xE, and CONS.C4xE. Note that CONS.C3xE is the parental CONS.C mutant presented in Fig. 3. Transformation by each isolate was quantitated as a percentage of p185neu. Results represent the average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as −, +, or ++ as described in Fig. 1. For transformation results shown in B, numerical values are also shown in parentheses as the percentage of p185neu.

Mentions: This issue was addressed through two different sets of mutants, one constructed in the background of CONS.A, [VVVEVVA], and another set in the background of CONS.C, [VVVEVVV]. These two sets of mutants yielded somewhat different results. As shown in Fig. 6 A, “singleGlu” derivatives of the CONS.A sequences were designed, each possessing only a single Glu residue located at different positions, and designated CONS.AE2, CONS.AE3, and CONS.AE4. None of these mutants was transforming, indicating that within the context of the CONS.A heptad repeat, a single-Glu residue is not sufficient to provide activation. This result is in contrast to the single Glu664 residue that leads to activation of p185neu, suggesting that there may be sequence information within the transmembrane domain, in addition to the Val664→ Glu mutation, that stabilizes dimerization (Cao et al., 1992). We considered the possibility that these mutant proteins might fail to reach the cell surface and that this defect might explain their inactivity in transformation assays. However, all of the single-Glu proteins were expressed at the cell surface, as shown by indirect immunofluorescence in Fig. 4, G and H, for CONS.AE2 and CONS.AE4.


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 number of Glu  residues. (A) Derivatives of CONS.A. Three  derivatives were constructed, each having a  single Glu residue. Depending upon the position of the Glu residue, these mutants were  designated CONS.AE2, CONS.AE3, and  CONS.AE4. (B) Derivatives of CONS.C. Derivatives were constructed in the background  of CONS.C having from one to four Glu residues. These clones are designated CONS.C1xE,  CONS.C2xE, CONS.C3xE, and CONS.C4xE.  Note that CONS.C3xE is the parental  CONS.C mutant presented in Fig. 3. Transformation by each isolate was quantitated as  a percentage of p185neu. Results represent the  average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as −, +, or ++ as described in Fig. 1. For transformation results  shown in B, numerical values are also shown in parentheses as the percentage of p185neu.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139875&req=5

Figure 6: Importance of the number of Glu residues. (A) Derivatives of CONS.A. Three derivatives were constructed, each having a single Glu residue. Depending upon the position of the Glu residue, these mutants were designated CONS.AE2, CONS.AE3, and CONS.AE4. (B) Derivatives of CONS.C. Derivatives were constructed in the background of CONS.C having from one to four Glu residues. These clones are designated CONS.C1xE, CONS.C2xE, CONS.C3xE, and CONS.C4xE. Note that CONS.C3xE is the parental CONS.C mutant presented in Fig. 3. Transformation by each isolate was quantitated as a percentage of p185neu. Results represent the average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as −, +, or ++ as described in Fig. 1. For transformation results shown in B, numerical values are also shown in parentheses as the percentage of p185neu.
Mentions: This issue was addressed through two different sets of mutants, one constructed in the background of CONS.A, [VVVEVVA], and another set in the background of CONS.C, [VVVEVVV]. These two sets of mutants yielded somewhat different results. As shown in Fig. 6 A, “singleGlu” derivatives of the CONS.A sequences were designed, each possessing only a single Glu residue located at different positions, and designated CONS.AE2, CONS.AE3, and CONS.AE4. None of these mutants was transforming, indicating that within the context of the CONS.A heptad repeat, a single-Glu residue is not sufficient to provide activation. This result is in contrast to the single Glu664 residue that leads to activation of p185neu, suggesting that there may be sequence information within the transmembrane domain, in addition to the Val664→ Glu mutation, that stabilizes dimerization (Cao et al., 1992). We considered the possibility that these mutant proteins might fail to reach the cell surface and that this defect might explain their inactivity in transformation assays. However, all of the single-Glu proteins were expressed at the cell surface, as shown by indirect immunofluorescence in Fig. 4, G and H, for CONS.AE2 and CONS.AE4.

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