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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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Isolates recovered from pools with degenerate transmembrane domains. (A) The sequences of wild-type p185c-neu  and oncogenic p185neu (with the mutation Val664ā†’ Glu) are  shown. The locations of the presumptive heptad repeats of the  transmembrane region are shown, along with the letters indicating the heptad positions. Vertical lines designate the probable  borders of the transmembrane domain. The activating Glu residue of p185neu is boldfaced. The five-residue sequence motif  found in many receptor tyrosine kinase transmembrane domains  (Sternberg and Gullick, 1990), referred to as P0-P4, corresponds  to residues 661ā€“665. The location of P0 and P3 are shown. (B)  Oligonucleotides coding for degenerate transmembrane domains  were synthesized, amplified, and cloned into pSV2neuNheI/SacI  as described in Materials and Methods. In the first pool of degenerate oligos, codons for either Ala, Val, Gly, or Glu were randomly targeted to the ā€œa,ā€ ā€œd,ā€ and ā€œeā€ positions. In Pool 2,  codons for Ala, Val, or Gly, but not Glu, were targeted to the  same positions. Asterisks denote the positions at which these degeneracies were targeted. (C) Pool 1 was transfected into  NIH3T3 cells; individual foci were expanded and used for PCR-  mediated recovery of the unique transmembrane domain present  in each focus. Each recovered transmembrane domain was ligated into pSV2neuNheI/SacI to ensure that transformation was  due to a unique transmembrane domain rather than a mixture of  sequences. The sequences of these transforming isolates are  shown, with dashes designating the residues that remained unchanged from the parent p185c-neu. Glu residues are boldfaced.  (D) For comparison, nontransforming isolates from Pool 1 were  also characterized. The sequences of some of these isolates are  shown. For A, C, and D, 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 follows: āˆ’, 0ā€“5%  of p185neu; +, 6ā€“40% of p185neu; ++ , 41ā€“100% of p185neu. For B,  transformation by DNA representing the entire pool was recorded as follows: āˆ’, 0 foci per 10 Ī¼g of DNA; ++, āˆ¼35 foci per  10 Ī¼g of DNA.
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Figure 1: Isolates recovered from pools with degenerate transmembrane domains. (A) The sequences of wild-type p185c-neu and oncogenic p185neu (with the mutation Val664ā†’ Glu) are shown. The locations of the presumptive heptad repeats of the transmembrane region are shown, along with the letters indicating the heptad positions. Vertical lines designate the probable borders of the transmembrane domain. The activating Glu residue of p185neu is boldfaced. The five-residue sequence motif found in many receptor tyrosine kinase transmembrane domains (Sternberg and Gullick, 1990), referred to as P0-P4, corresponds to residues 661ā€“665. The location of P0 and P3 are shown. (B) Oligonucleotides coding for degenerate transmembrane domains were synthesized, amplified, and cloned into pSV2neuNheI/SacI as described in Materials and Methods. In the first pool of degenerate oligos, codons for either Ala, Val, Gly, or Glu were randomly targeted to the ā€œa,ā€ ā€œd,ā€ and ā€œeā€ positions. In Pool 2, codons for Ala, Val, or Gly, but not Glu, were targeted to the same positions. Asterisks denote the positions at which these degeneracies were targeted. (C) Pool 1 was transfected into NIH3T3 cells; individual foci were expanded and used for PCR- mediated recovery of the unique transmembrane domain present in each focus. Each recovered transmembrane domain was ligated into pSV2neuNheI/SacI to ensure that transformation was due to a unique transmembrane domain rather than a mixture of sequences. The sequences of these transforming isolates are shown, with dashes designating the residues that remained unchanged from the parent p185c-neu. Glu residues are boldfaced. (D) For comparison, nontransforming isolates from Pool 1 were also characterized. The sequences of some of these isolates are shown. For A, C, and D, 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 follows: āˆ’, 0ā€“5% of p185neu; +, 6ā€“40% of p185neu; ++ , 41ā€“100% of p185neu. For B, transformation by DNA representing the entire pool was recorded as follows: āˆ’, 0 foci per 10 Ī¼g of DNA; ++, āˆ¼35 foci per 10 Ī¼g of DNA.

Mentions: Oligonucleotides were synthesized encoding the p185c-neu transmembrane domain with degenerate codons targeted to the presumptive Ī±-helical positions designated ā€œa,ā€ ā€œd,ā€ and ā€œe,ā€ shown in Fig. 1. In Pool 1, codons for Val, Ala, Gly, and Glu were targeted to each of these positions. In Pool 2, the codons for Val, Ala, or Gly, but not Glu, were targeted to these positions. The degenerate oligonucleotides were synthesized on an oligonucleotide synthesizer (Appiled Biosystems Inc., Foster City, CA) and then amplified by PCR. The degenerate pools were ligated into a pSV2-derived vector encoding p185c-neu (pSV2neuNheI/SacI), replacing the wild-type transmembrane domain at silent NheI and SacI restriction sites (Webster and Donoghue, 1996). The NheI site corresponds to bases 1973ā€“1978, and the SacI site corresponds to bases 2114ā€“2119, in the published nucleotide sequence encoding p185c-neu (Bargmann et al., 1986a).


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)

Isolates recovered from pools with degenerate transmembrane domains. (A) The sequences of wild-type p185c-neu  and oncogenic p185neu (with the mutation Val664ā†’ Glu) are  shown. The locations of the presumptive heptad repeats of the  transmembrane region are shown, along with the letters indicating the heptad positions. Vertical lines designate the probable  borders of the transmembrane domain. The activating Glu residue of p185neu is boldfaced. The five-residue sequence motif  found in many receptor tyrosine kinase transmembrane domains  (Sternberg and Gullick, 1990), referred to as P0-P4, corresponds  to residues 661ā€“665. The location of P0 and P3 are shown. (B)  Oligonucleotides coding for degenerate transmembrane domains  were synthesized, amplified, and cloned into pSV2neuNheI/SacI  as described in Materials and Methods. In the first pool of degenerate oligos, codons for either Ala, Val, Gly, or Glu were randomly targeted to the ā€œa,ā€ ā€œd,ā€ and ā€œeā€ positions. In Pool 2,  codons for Ala, Val, or Gly, but not Glu, were targeted to the  same positions. Asterisks denote the positions at which these degeneracies were targeted. (C) Pool 1 was transfected into  NIH3T3 cells; individual foci were expanded and used for PCR-  mediated recovery of the unique transmembrane domain present  in each focus. Each recovered transmembrane domain was ligated into pSV2neuNheI/SacI to ensure that transformation was  due to a unique transmembrane domain rather than a mixture of  sequences. The sequences of these transforming isolates are  shown, with dashes designating the residues that remained unchanged from the parent p185c-neu. Glu residues are boldfaced.  (D) For comparison, nontransforming isolates from Pool 1 were  also characterized. The sequences of some of these isolates are  shown. For A, C, and D, 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 follows: āˆ’, 0ā€“5%  of p185neu; +, 6ā€“40% of p185neu; ++ , 41ā€“100% of p185neu. For B,  transformation by DNA representing the entire pool was recorded as follows: āˆ’, 0 foci per 10 Ī¼g of DNA; ++, āˆ¼35 foci per  10 Ī¼g of DNA.
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Related In: Results  -  Collection

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Figure 1: Isolates recovered from pools with degenerate transmembrane domains. (A) The sequences of wild-type p185c-neu and oncogenic p185neu (with the mutation Val664ā†’ Glu) are shown. The locations of the presumptive heptad repeats of the transmembrane region are shown, along with the letters indicating the heptad positions. Vertical lines designate the probable borders of the transmembrane domain. The activating Glu residue of p185neu is boldfaced. The five-residue sequence motif found in many receptor tyrosine kinase transmembrane domains (Sternberg and Gullick, 1990), referred to as P0-P4, corresponds to residues 661ā€“665. The location of P0 and P3 are shown. (B) Oligonucleotides coding for degenerate transmembrane domains were synthesized, amplified, and cloned into pSV2neuNheI/SacI as described in Materials and Methods. In the first pool of degenerate oligos, codons for either Ala, Val, Gly, or Glu were randomly targeted to the ā€œa,ā€ ā€œd,ā€ and ā€œeā€ positions. In Pool 2, codons for Ala, Val, or Gly, but not Glu, were targeted to the same positions. Asterisks denote the positions at which these degeneracies were targeted. (C) Pool 1 was transfected into NIH3T3 cells; individual foci were expanded and used for PCR- mediated recovery of the unique transmembrane domain present in each focus. Each recovered transmembrane domain was ligated into pSV2neuNheI/SacI to ensure that transformation was due to a unique transmembrane domain rather than a mixture of sequences. The sequences of these transforming isolates are shown, with dashes designating the residues that remained unchanged from the parent p185c-neu. Glu residues are boldfaced. (D) For comparison, nontransforming isolates from Pool 1 were also characterized. The sequences of some of these isolates are shown. For A, C, and D, 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 follows: āˆ’, 0ā€“5% of p185neu; +, 6ā€“40% of p185neu; ++ , 41ā€“100% of p185neu. For B, transformation by DNA representing the entire pool was recorded as follows: āˆ’, 0 foci per 10 Ī¼g of DNA; ++, āˆ¼35 foci per 10 Ī¼g of DNA.
Mentions: Oligonucleotides were synthesized encoding the p185c-neu transmembrane domain with degenerate codons targeted to the presumptive Ī±-helical positions designated ā€œa,ā€ ā€œd,ā€ and ā€œe,ā€ shown in Fig. 1. In Pool 1, codons for Val, Ala, Gly, and Glu were targeted to each of these positions. In Pool 2, the codons for Val, Ala, or Gly, but not Glu, were targeted to these positions. The degenerate oligonucleotides were synthesized on an oligonucleotide synthesizer (Appiled Biosystems Inc., Foster City, CA) and then amplified by PCR. The degenerate pools were ligated into a pSV2-derived vector encoding p185c-neu (pSV2neuNheI/SacI), replacing the wild-type transmembrane domain at silent NheI and SacI restriction sites (Webster and Donoghue, 1996). The NheI site corresponds to bases 1973ā€“1978, and the SacI site corresponds to bases 2114ā€“2119, in the published nucleotide sequence encoding p185c-neu (Bargmann et al., 1986a).

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