| Abstract: | Association of 125I-nerve growth factor (NGF) with PC12 pheochromocytoma cells was studied. Surface-bound and internalized NGF were distinguished by differential release of the former at low pH, high salt. Binding to the surface was rapid; at 0.2 nM (5 ng/ml) 125I-NGF, this was near-maximal within 5 min. Internalization, in contrast, did not start until about 2 min after NGF exposure and, thereafter, proceeded linearly for at least 1/2-1 h. By the latter time, approximately 75% of total bound NGF was within rather than on the surface of the cells. Binding versus concentration experiments indicated two distinct classes of surface binding sites. For both naive cells and cells treated with NGF for at least a week (primed cells), about 7% of the receptors had an apparent binding constant of about 0.3 nM; the remaining sites half-saturated at approximately 4 nM NGF. The number of each type of site was 3--4-fold higher/mg of protein in primed cells. For both naive and primed cultures, internalization appeared to be mediated by a single class of uptake sites which half-saturated at about 0.3 nM. The maximal rate of uptake by primed cells (200 fmol/h/mg protein) was about twice that for naive cells. Light and electron microscopic autoradiography indicated that the density of binding was substantially higher in primed cultures and that this increase took place over a time course of days to weeks. These findings suggest that NGF brings about long-term increases in its own high- and low-affinity surface receptors, but is internalized only via the high-affinity sites. |
