Nerve growth factor signals via preexisting TrkA receptor oligomers.
Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.
Department of Pathology and Laboratory Medicine, UCLA School of Medicine, University of California, Los Angeles, California 90095-1732 USA. pmischel@mednet.ucla.edu
Paul S
PS
Mischel
Joy A
JA
Umbach
Sepehr
S
Eskandari
Shane G
SG
Smith
Cameron B
CB
Gundersen
Guido A
GA
Zampighi
D000818:Animals; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003609:Dactinomycin / Q000494:pharmacology; D019281:Dimerization; D004594:Electrophysiology; D005614:Freeze Fracturing; D015151:Immunoblotting; D008854:Microscopy, Electron; D020932:Nerve Growth Factor / Q000378:metabolism*; D011485:Protein Binding; D018075:RNA, Complementary / Q000378:metabolism; D020917:Receptor, trkA / Q000378:metabolism*; D015398:Signal Transduction; D014982:Xenopus laevis
D018075:RNA, Complementary; D003609:Dactinomycin; D020932:Nerve Growth Factor; D020917:Receptor, trkA