Long term association of the cytokine receptor gp130 and the Janus kinase Jak1 revealed by FRAP analysis.
Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow (YFP) or cyan (CFP) fluorescent protein, were found to be colocalized predominantly at intracellular vesicular structures and at the plasma membrane. Quantitative fluorescence recovery after photobleaching (FRAP) analysis at the plasma membrane revealed equal mobilities for gp130-YFP and Jak1-YFP. Thus, Jak1-YFP diffuses like a transmembrane protein indicating that membrane-bound Jak1 does not exchange rapidly with cytosolic Jaks. Applying a novel dual-color FRAP approach we found that immobilization of gp130-CFP by a pair of monoclonal antibodies led to a corresponding immobilization of co-transfected Jak1-YFP. We conclude from these findings that Jak1, once bound to a gp130 molecule, does not exchange between different receptors at the plasma membrane neither via the cytoplasmic compartment nor via a membrane-associated state.
Giese
Bernd
B
Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany.
Au-Yeung
Chi-King
CK
Herrmann
Andreas
A
Diefenbach
Sandra
S
Haan
Claude
C
Küster
Andrea
A
Wortmann
Saskia B
SB
Roderburg
Christoph
C
Heinrich
Peter C
PC
Behrmann
Iris
I
Müller-Newen
Gerhard
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D050822:Cytokine Receptor gp130; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D005822:Genetic Vectors; D053613:Janus Kinase 1; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130; D011505:Protein-Tyrosine Kinases; D053613:Janus Kinase 1