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Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

Tertoolen LG, Blanchetot C, Jiang G, Overvoorde J, Gadella TW, Hunter T, den Hertog J - BMC Cell Biol. (2001)

Bottom Line: RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker.Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands. leon@niob.knaw.nl

ABSTRACT

Background: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.

Results: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPalpha, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPalpha -CFP and -YFP fusion proteins, and thus that RPTPalpha dimerized in living cells. RPTPalpha dimerization was constitutive, extensive and specific. RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.

Conclusions: We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

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Homodimerization of RPTPα as assessed using a chemical cross-linker. (A) Distinct RPTPα constructs with different molecular weights were used to be able to discriminate between them. The long form (L) is a full length RPTPα construct with YFP fused to the C-terminus, while the short form (S) lacks almost all cytoplasmic sequences and contains CFP fused to residue 200, close to the transmembrane domain. (B) SK-N-MC cells were transiently transfected with the constructs depicted in (A). The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate (BS3), or left untreated, as indicated. Aliquots of the total cell lysates were run on a 5% SDS-polyacrylamide gel, the gel was blotted, and the immunoblot was probed with anti-HA-tag antibody. The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left (in kDa). The position of monomeric RPTPα-YFP (the long form, L), dimeric RPTPα-YFP (LL), monomeric RPTPα-200-CFP (the short form, S), dimeric RPTPα-200-CFP (SS), and of the RPTPα-YFP/ RPTPα-200-CFP heterodimer (LS) are indicated, as well as a non-specific band (NS). Lysates from BS3-treated cells that had been transfected with either RPTPα-YFP or RPTPα-200-CFP were mixed (mix), clearly demonstrating that the heterodimer (LS) only formed in cells that had been co-transfected with RPTPα-YFP and RPTPα-200-CFP.
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Figure 4: Homodimerization of RPTPα as assessed using a chemical cross-linker. (A) Distinct RPTPα constructs with different molecular weights were used to be able to discriminate between them. The long form (L) is a full length RPTPα construct with YFP fused to the C-terminus, while the short form (S) lacks almost all cytoplasmic sequences and contains CFP fused to residue 200, close to the transmembrane domain. (B) SK-N-MC cells were transiently transfected with the constructs depicted in (A). The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate (BS3), or left untreated, as indicated. Aliquots of the total cell lysates were run on a 5% SDS-polyacrylamide gel, the gel was blotted, and the immunoblot was probed with anti-HA-tag antibody. The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left (in kDa). The position of monomeric RPTPα-YFP (the long form, L), dimeric RPTPα-YFP (LL), monomeric RPTPα-200-CFP (the short form, S), dimeric RPTPα-200-CFP (SS), and of the RPTPα-YFP/ RPTPα-200-CFP heterodimer (LS) are indicated, as well as a non-specific band (NS). Lysates from BS3-treated cells that had been transfected with either RPTPα-YFP or RPTPα-200-CFP were mixed (mix), clearly demonstrating that the heterodimer (LS) only formed in cells that had been co-transfected with RPTPα-YFP and RPTPα-200-CFP.

Mentions: We used the non-cell-permeable irreversible chemical cross-linker, bis- [sulfosuccinimidyl]suberate (BS3), on intact cells to confirm that RPTPα-YFP dimerized. A considerable proportion of RPTPα-YFP, designated long form (L) (Fig. 4A), migrated with an apparent molecular weight that was consistent with dimerization following treatment of the cells with BS3 (indicated by LL) (Fig. 4B). The proportion of cross-linked dimers appeared to be low. It is noteworthy that - similar to RPTPα-YFP - less than 10% of the constitutively dimeric RPTPα-P137C was cross-linked by BS3 (data not shown), suggesting that the efficiency of cross-linking was low under these conditions. These results are similar to previously published results with wild type RPTPα, lacking GFP derivatives [24], indicating that the GFP-derivatives neither enhanced nor interfered with RPTPα-YFP dimerization. We tested dimerization of a deletion mutant of RPTPα, enabling us to investigate whether RPTPα homodimerized, or merely interacted with another protein of similar size. RPTPα-200-CFP, lacking almost the entire cytoplasmic domain, including the wedge, had an apparent molecular weight of approximately 110 kDa, and was designated short form (S) (Fig. 4A). RPTPα-200-CFP migrated as a dimer following treatment of the cells with BS3 (indicated by SS) (Fig. 4B). Dimerization of the short form appeared to be more efficient than of the long form (Fig. 4B). However, we cannot exclude that this is due - in part - to an artefact as a result of more efficient blotting of smaller proteins. Co-transfection of RPTPα-YFP (L) and RPTPα-200-CFP (S) followed by treatment with BS3 led to the formation of LL-dimers, SS-dimers, and a novel intermediate form, LS-heterodimers. Mixing of lysates from BS3-treated cells transfected with only RPTPα-YFP or RPTPα-200-CFP illustrated that the LS heterodimer was only detected after BS3-treatment of cells co-expressing RPTPα-YFP and RPTPα-200-CFP (Fig. 4B). Thus, BS3-mediated cross-linking confirmed the FRET measurements, and the heterodimerization results of the long and short forms demonstrated that RPTPα homodimerized specifically.


Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

Tertoolen LG, Blanchetot C, Jiang G, Overvoorde J, Gadella TW, Hunter T, den Hertog J - BMC Cell Biol. (2001)

Homodimerization of RPTPα as assessed using a chemical cross-linker. (A) Distinct RPTPα constructs with different molecular weights were used to be able to discriminate between them. The long form (L) is a full length RPTPα construct with YFP fused to the C-terminus, while the short form (S) lacks almost all cytoplasmic sequences and contains CFP fused to residue 200, close to the transmembrane domain. (B) SK-N-MC cells were transiently transfected with the constructs depicted in (A). The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate (BS3), or left untreated, as indicated. Aliquots of the total cell lysates were run on a 5% SDS-polyacrylamide gel, the gel was blotted, and the immunoblot was probed with anti-HA-tag antibody. The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left (in kDa). The position of monomeric RPTPα-YFP (the long form, L), dimeric RPTPα-YFP (LL), monomeric RPTPα-200-CFP (the short form, S), dimeric RPTPα-200-CFP (SS), and of the RPTPα-YFP/ RPTPα-200-CFP heterodimer (LS) are indicated, as well as a non-specific band (NS). Lysates from BS3-treated cells that had been transfected with either RPTPα-YFP or RPTPα-200-CFP were mixed (mix), clearly demonstrating that the heterodimer (LS) only formed in cells that had been co-transfected with RPTPα-YFP and RPTPα-200-CFP.
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Figure 4: Homodimerization of RPTPα as assessed using a chemical cross-linker. (A) Distinct RPTPα constructs with different molecular weights were used to be able to discriminate between them. The long form (L) is a full length RPTPα construct with YFP fused to the C-terminus, while the short form (S) lacks almost all cytoplasmic sequences and contains CFP fused to residue 200, close to the transmembrane domain. (B) SK-N-MC cells were transiently transfected with the constructs depicted in (A). The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate (BS3), or left untreated, as indicated. Aliquots of the total cell lysates were run on a 5% SDS-polyacrylamide gel, the gel was blotted, and the immunoblot was probed with anti-HA-tag antibody. The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left (in kDa). The position of monomeric RPTPα-YFP (the long form, L), dimeric RPTPα-YFP (LL), monomeric RPTPα-200-CFP (the short form, S), dimeric RPTPα-200-CFP (SS), and of the RPTPα-YFP/ RPTPα-200-CFP heterodimer (LS) are indicated, as well as a non-specific band (NS). Lysates from BS3-treated cells that had been transfected with either RPTPα-YFP or RPTPα-200-CFP were mixed (mix), clearly demonstrating that the heterodimer (LS) only formed in cells that had been co-transfected with RPTPα-YFP and RPTPα-200-CFP.
Mentions: We used the non-cell-permeable irreversible chemical cross-linker, bis- [sulfosuccinimidyl]suberate (BS3), on intact cells to confirm that RPTPα-YFP dimerized. A considerable proportion of RPTPα-YFP, designated long form (L) (Fig. 4A), migrated with an apparent molecular weight that was consistent with dimerization following treatment of the cells with BS3 (indicated by LL) (Fig. 4B). The proportion of cross-linked dimers appeared to be low. It is noteworthy that - similar to RPTPα-YFP - less than 10% of the constitutively dimeric RPTPα-P137C was cross-linked by BS3 (data not shown), suggesting that the efficiency of cross-linking was low under these conditions. These results are similar to previously published results with wild type RPTPα, lacking GFP derivatives [24], indicating that the GFP-derivatives neither enhanced nor interfered with RPTPα-YFP dimerization. We tested dimerization of a deletion mutant of RPTPα, enabling us to investigate whether RPTPα homodimerized, or merely interacted with another protein of similar size. RPTPα-200-CFP, lacking almost the entire cytoplasmic domain, including the wedge, had an apparent molecular weight of approximately 110 kDa, and was designated short form (S) (Fig. 4A). RPTPα-200-CFP migrated as a dimer following treatment of the cells with BS3 (indicated by SS) (Fig. 4B). Dimerization of the short form appeared to be more efficient than of the long form (Fig. 4B). However, we cannot exclude that this is due - in part - to an artefact as a result of more efficient blotting of smaller proteins. Co-transfection of RPTPα-YFP (L) and RPTPα-200-CFP (S) followed by treatment with BS3 led to the formation of LL-dimers, SS-dimers, and a novel intermediate form, LS-heterodimers. Mixing of lysates from BS3-treated cells transfected with only RPTPα-YFP or RPTPα-200-CFP illustrated that the LS heterodimer was only detected after BS3-treatment of cells co-expressing RPTPα-YFP and RPTPα-200-CFP (Fig. 4B). Thus, BS3-mediated cross-linking confirmed the FRET measurements, and the heterodimerization results of the long and short forms demonstrated that RPTPα homodimerized specifically.

Bottom Line: RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker.Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands. leon@niob.knaw.nl

ABSTRACT

Background: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.

Results: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPalpha, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPalpha -CFP and -YFP fusion proteins, and thus that RPTPalpha dimerized in living cells. RPTPalpha dimerization was constitutive, extensive and specific. RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.

Conclusions: We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Show MeSH