Molecular characteristics of nerve growth factor receptors on PC12 cells.

M Hosang,E M Shooter

doi:10.1016/s0021-9258(18)89782-4

Abstract

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.

Highlights

Cross-linking of 12'I-nerve growth factor (NGF) to NGF' is polypeptide hormone which is involved in the PC12 cells with the photoreactive heterobifunctional development and maintenance of several tissues of neural agent N-hydroxysuccinimidyl-4-azidobenzoateresults crest origin including the sympathetic nervous system (1-4)
12'I-NGF to and cross-linking into all these species is abolished in thepresence of excess unlabeled NGF but not in thepresence of unlabeled epidermalgrowth factor, insulin, or bovine pancreatic trypsin inhibitor
When PC12 cells with bound lZ6I-NGFare incubated in excess unlabeled NGF at 0 "C prior to cross-linking, only the M, 158,000species remains

Summary

Mr x

The apparent molecular weights and the relative amounts of the two labeled species did not change when the HSAR concentration was varied over a 5000-fold range, andthe absolute labeling intensity of the individual bands was decreased only moderately at the lowest HSAR concentration used, 10 nM (data not shown). Loaded cells with excess unlabeled NGF was performed for 30 min at 37 "C instead, as shown in Fig. 4 A , lane 3, labeling of the M r 158,000 component was abolished in line with the common proteaseinhibitors.Inordertoexaminewhether such proteases were responsible for the labeling pattern ob-. This band may reflect covalent lane 2) This result identifies the M,105,000 species as the cross-linking of the receptor components in the M,100,000 low-affinity NGF receptor. This suggests that, as with PC12 and M,158,000complexesto one another or to a third protein, cells, the lower-molecular-weight species is the cross-linked or it maybe yet another receptor component which is not low-affinity (site 11) receptor complex, and the high-molecu- labeled efficiently.

DISCUSSION

Considerable evidence is accumulating that at least some