Interaction with TrkA immobilizes gp75 in the high affinity nerve growth factor receptor complex.
It has been proposed that the high affinity nerve growth factor (NGF) receptor required for NGF response is a complex of two receptor proteins, gp75 and the tyrosine kinase TrkA, but direct biochemical or biophysical evidence has been lacking. We have previously shown using fluorescence recovery after photobleaching that gp75 is highly mobile on NGF-nonresponsive cells, but relatively immobile on NGF-responsive cells. In this report, we show that a physical interaction with TrkA causes gp75 immobilization. We found that gp75 is relatively mobile on TrkA negative nnr5 cells, a PC12 variant which is nonresponsive to NGF. In contrast, on T14 nnr5 cells (which bear a TrkA expression vector) gp75 is relatively immobile. Similarly, using baculoviruses to express gp75 and TrkA on Sf9 insect cells, we found that TrkA immobilizes gp75 molecules. The related receptor, TrkB, caused a more modest immobilization of gp75. Immobilization was found to require intact TrkA kinase and gp75 cytoplasmic domains, paralleling the requirements of high affinity binding of NGF. Analysis of gp75 diffusion coefficients indicates that mutated gp75 and TrkA molecules may form a complex, even in the absence of the ability to bind NGF with high affinity.
Wolf
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DE
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
McKinnon
C A
CA
Daou
M C
MC
Stephens
R M
RM
Kaplan
D R
DR
Ross
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AH
D000818:Animals; D001665:Binding Sites; D004058:Diffusion; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D008560:Membrane Fluidity; D009036:Moths; D009414:Nerve Growth Factors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D011940:Receptor Aggregation; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D020801:Receptor, Ciliary Neurotrophic Factor; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA