Limits...
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.

Show MeSH

Related in: MedlinePlus

Indirect immunofluorescence of consensus mutants and derivatives. Indirect  immunofluorescence using the  monoclonal antibody 7.16.4 directed against the extracellular  region of p185c-neu and a fluorescein-conjugated goat anti– mouse secondary antibody revealed both cell surface and  intracellular protein expression. A–L show nonpermeabilized cells to examine cell surface expression. (A) p185c-neu;  (B) p185neu; (C) CONS.A; (D)  CONS.B; (E) CONS.C; (F)  CONS.D; (G) CONS.AE2; (H)  CONS.AE4; (I) CONS.AE→ K;  (J) CONS.AE→ Q; (K) CONS.  AE→ S; (L) CONS.CE→ V. M–O  show cells after permeabilization to examine intracellular  expression. (M) p185c-neu; (N)  p185neu; (O) CONS.AE→ D.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139875&req=5

Figure 4: Indirect immunofluorescence of consensus mutants and derivatives. Indirect immunofluorescence using the monoclonal antibody 7.16.4 directed against the extracellular region of p185c-neu and a fluorescein-conjugated goat anti– mouse secondary antibody revealed both cell surface and intracellular protein expression. A–L show nonpermeabilized cells to examine cell surface expression. (A) p185c-neu; (B) p185neu; (C) CONS.A; (D) CONS.B; (E) CONS.C; (F) CONS.D; (G) CONS.AE2; (H) CONS.AE4; (I) CONS.AE→ K; (J) CONS.AE→ Q; (K) CONS. AE→ S; (L) CONS.CE→ V. M–O show cells after permeabilization to examine intracellular expression. (M) p185c-neu; (N) p185neu; (O) CONS.AE→ D.

Mentions: As shown in Fig. 4, A–L, indirect immunofluorescence demonstrated that most of the mutants exhibited cell surface expression. The ability of these altered receptors to reach the surface indicates that the mutant transmembrane domains did not interfere with the proper localization of the protein. The only exception was CONS.AE→ D, which exhibited little or no cell surface staining, even though the protein is clearly being translated, as shown by the intracellular staining of permeabilized cells (Fig. 4, O). However, its reduced surface expression may be due to aggregation or interaction with another protein that is retained in the ER and/or Golgi, as discussed previously for some of the nontransforming mutants, DEG.7, DEG.9, and DEG.10.


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)

Indirect immunofluorescence of consensus mutants and derivatives. Indirect  immunofluorescence using the  monoclonal antibody 7.16.4 directed against the extracellular  region of p185c-neu and a fluorescein-conjugated goat anti– mouse secondary antibody revealed both cell surface and  intracellular protein expression. A–L show nonpermeabilized cells to examine cell surface expression. (A) p185c-neu;  (B) p185neu; (C) CONS.A; (D)  CONS.B; (E) CONS.C; (F)  CONS.D; (G) CONS.AE2; (H)  CONS.AE4; (I) CONS.AE→ K;  (J) CONS.AE→ Q; (K) CONS.  AE→ S; (L) CONS.CE→ V. M–O  show cells after permeabilization to examine intracellular  expression. (M) p185c-neu; (N)  p185neu; (O) CONS.AE→ D.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Indirect immunofluorescence of consensus mutants and derivatives. Indirect immunofluorescence using the monoclonal antibody 7.16.4 directed against the extracellular region of p185c-neu and a fluorescein-conjugated goat anti– mouse secondary antibody revealed both cell surface and intracellular protein expression. A–L show nonpermeabilized cells to examine cell surface expression. (A) p185c-neu; (B) p185neu; (C) CONS.A; (D) CONS.B; (E) CONS.C; (F) CONS.D; (G) CONS.AE2; (H) CONS.AE4; (I) CONS.AE→ K; (J) CONS.AE→ Q; (K) CONS. AE→ S; (L) CONS.CE→ V. M–O show cells after permeabilization to examine intracellular expression. (M) p185c-neu; (N) p185neu; (O) CONS.AE→ D.
Mentions: As shown in Fig. 4, A–L, indirect immunofluorescence demonstrated that most of the mutants exhibited cell surface expression. The ability of these altered receptors to reach the surface indicates that the mutant transmembrane domains did not interfere with the proper localization of the protein. The only exception was CONS.AE→ D, which exhibited little or no cell surface staining, even though the protein is clearly being translated, as shown by the intracellular staining of permeabilized cells (Fig. 4, O). However, its reduced surface expression may be due to aggregation or interaction with another protein that is retained in the ER and/or Golgi, as discussed previously for some of the nontransforming mutants, DEG.7, DEG.9, and DEG.10.

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