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

Consensus sequences and derivatives. (A) The p185c-neu and p185neu sequences  are shown. The locations of the presumptive  heptad repeats, along with the letters indicating the heptad positions, are specified. Vertical lines designate the probable borders of  the transmembrane domain. Glu664 is in boldface. (B) Consensus sequence mutants were  constructed as described in the text. The  placement of the Glu residues in each mutant  is identical. Based on the variations found in  the transforming isolates from degenerate  Pool 1, the heptad position g in CONS.B and  CONS.C was mutated from Ala in CONS.A  to Gly and Val, respectively. The repeating  heptad in CONS.D is heptad 2 from p185neu  with Thr662 changed to Val. The mutant  CONS.CE→ V has a transmembrane domain  composed entirely of Val residues and was  constructed as a negative control. Glu residues are boldfaced. (C) Derivatives of  CONS.A mutant. Derivatives were designed  in which the Glu residues were substituted by  other residues capable of hydrogen bonding,  such as Lys, Gln, Ser, or Asp. For example,  CONS.AE→ Q is identical to CONS.A except that the Glu residues are substituted by Gln. 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. Surface expression was determined by indirect immunofluorescence, as described in text.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139875&req=5

Figure 3: Consensus sequences and derivatives. (A) The p185c-neu and p185neu sequences are shown. The locations of the presumptive heptad repeats, along with the letters indicating the heptad positions, are specified. Vertical lines designate the probable borders of the transmembrane domain. Glu664 is in boldface. (B) Consensus sequence mutants were constructed as described in the text. The placement of the Glu residues in each mutant is identical. Based on the variations found in the transforming isolates from degenerate Pool 1, the heptad position g in CONS.B and CONS.C was mutated from Ala in CONS.A to Gly and Val, respectively. The repeating heptad in CONS.D is heptad 2 from p185neu with Thr662 changed to Val. The mutant CONS.CE→ V has a transmembrane domain composed entirely of Val residues and was constructed as a negative control. Glu residues are boldfaced. (C) Derivatives of CONS.A mutant. Derivatives were designed in which the Glu residues were substituted by other residues capable of hydrogen bonding, such as Lys, Gln, Ser, or Asp. For example, CONS.AE→ Q is identical to CONS.A except that the Glu residues are substituted by Gln. 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. Surface expression was determined by indirect immunofluorescence, as described in text.

Mentions: The transmembrane domains of the transforming mutants DEG.1–DEG.5 suggested that there are many allowed positions for Glu residues, yet the distinction between these sequences and those of the nontransforming mutants DEG.6–DEG.10 did not suggest clear rules for predicting which specific sequences will result in receptor activation and transformation. We therefore designed simple consensus repeats, containing a centrally located Glu residue, with most of the other residues substituted by Val, such as [VVVEVVA]n, [VVVEVVG]n, [VVVEVVV]n, or [AVVEGVL]n (designated CONS.A, CONS.B, CONS.C, and CONS.D, respectively). Mutants were then constructed with the transmembrane domain composed entirely of these consensus sequences, repeated over the entire transmembrane domain of ∼25 residues. Surprisingly, these constructs were transforming, with the CONS.A and CONS.D constructs exhibiting the greatest activity (Fig. 3 B). These results suggest that these simple repeating sequences contain all the information necessary for receptor activation. Evidently, there is little sequence specificity necessary for constitutive activation within the p185neu transmembrane domain itself.


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)

Consensus sequences and derivatives. (A) The p185c-neu and p185neu sequences  are shown. The locations of the presumptive  heptad repeats, along with the letters indicating the heptad positions, are specified. Vertical lines designate the probable borders of  the transmembrane domain. Glu664 is in boldface. (B) Consensus sequence mutants were  constructed as described in the text. The  placement of the Glu residues in each mutant  is identical. Based on the variations found in  the transforming isolates from degenerate  Pool 1, the heptad position g in CONS.B and  CONS.C was mutated from Ala in CONS.A  to Gly and Val, respectively. The repeating  heptad in CONS.D is heptad 2 from p185neu  with Thr662 changed to Val. The mutant  CONS.CE→ V has a transmembrane domain  composed entirely of Val residues and was  constructed as a negative control. Glu residues are boldfaced. (C) Derivatives of  CONS.A mutant. Derivatives were designed  in which the Glu residues were substituted by  other residues capable of hydrogen bonding,  such as Lys, Gln, Ser, or Asp. For example,  CONS.AE→ Q is identical to CONS.A except that the Glu residues are substituted by Gln. 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. Surface expression was determined by indirect immunofluorescence, as described in text.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Consensus sequences and derivatives. (A) The p185c-neu and p185neu sequences are shown. The locations of the presumptive heptad repeats, along with the letters indicating the heptad positions, are specified. Vertical lines designate the probable borders of the transmembrane domain. Glu664 is in boldface. (B) Consensus sequence mutants were constructed as described in the text. The placement of the Glu residues in each mutant is identical. Based on the variations found in the transforming isolates from degenerate Pool 1, the heptad position g in CONS.B and CONS.C was mutated from Ala in CONS.A to Gly and Val, respectively. The repeating heptad in CONS.D is heptad 2 from p185neu with Thr662 changed to Val. The mutant CONS.CE→ V has a transmembrane domain composed entirely of Val residues and was constructed as a negative control. Glu residues are boldfaced. (C) Derivatives of CONS.A mutant. Derivatives were designed in which the Glu residues were substituted by other residues capable of hydrogen bonding, such as Lys, Gln, Ser, or Asp. For example, CONS.AE→ Q is identical to CONS.A except that the Glu residues are substituted by Gln. 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. Surface expression was determined by indirect immunofluorescence, as described in text.
Mentions: The transmembrane domains of the transforming mutants DEG.1–DEG.5 suggested that there are many allowed positions for Glu residues, yet the distinction between these sequences and those of the nontransforming mutants DEG.6–DEG.10 did not suggest clear rules for predicting which specific sequences will result in receptor activation and transformation. We therefore designed simple consensus repeats, containing a centrally located Glu residue, with most of the other residues substituted by Val, such as [VVVEVVA]n, [VVVEVVG]n, [VVVEVVV]n, or [AVVEGVL]n (designated CONS.A, CONS.B, CONS.C, and CONS.D, respectively). Mutants were then constructed with the transmembrane domain composed entirely of these consensus sequences, repeated over the entire transmembrane domain of ∼25 residues. Surprisingly, these constructs were transforming, with the CONS.A and CONS.D constructs exhibiting the greatest activity (Fig. 3 B). These results suggest that these simple repeating sequences contain all the information necessary for receptor activation. Evidently, there is little sequence specificity necessary for constitutive activation within the p185neu transmembrane domain itself.

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