Abstract
Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.
Highlights
Tiate transmembrane signaling, an increasing number of receptor-tyrosine kinases have been found to be co-expressed with heterologous co-receptors lacking intrinsic kinase activity
Examples of such heteromeric receptors include the receptors for vascular endothelial growth factor, the flt-1 receptor-tyrosine kinase and neuropilin-1 [1]; the receptors for the glial-derived neurotrophic growth factor (GDNF), the ret receptor-tyrosine kinase and GRF␣ [2]; and the receptors for nerve growth factor (NGF), the Trk A receptor-tyrosine kinase and the p75 neurotrophin receptor [3]
In prior studies using PC12 cells, we had noted that expression of Trk A:p75 at ratios of 1.0:0.8 yielded a higher percentage of high affinity sites as compared with PC12 cells with a 1:20 ratio of Trk A:p75 [42]
Summary
Tiate transmembrane signaling, an increasing number of receptor-tyrosine kinases have been found to be co-expressed with heterologous co-receptors lacking intrinsic kinase activity. With the vascular endothelial growth factor and NGF ligands, co-expression of their receptor-tyrosine kinase and heterologous co-receptor generates receptor complexes that exhibit higher affinity binding constants than those exhibited by homodimeric receptor-tyrosine kinase complexes. A second, conformational model predicts that co-expression of both receptortyrosine kinase and co-receptor alters the conformation of the receptor-tyrosine kinase through allosteric interactions, generating a higher affinity binding site by altering the association or dissociation constants of the ligand with its receptor-tyrosine kinase. When Trk A receptors are co-expressed with p75, the rate of association is accelerated 25-fold, generating a new kinetic site exhibiting high affinity binding properties [7]. The ability of each receptor subunit to modulate the signaling cascades initiated by the co-receptor suggests that the receptors directly interact
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