Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.
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.
Tertoolen
L G
LG
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands. leon@niob.knaw.nl
Blanchetot
C
C
Jiang
G
G
Overvoorde
J
J
Gadella
T W
TW
Hunter
T
T
den Hertog
J
J
D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004735:Energy Transfer; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017027:Protein Tyrosine Phosphatases; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4