A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Abstract

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound ¹²⁵I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: K_D = 5.2 nM sites and K_D = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and ¹²⁵I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (K_D = 1.0 nM), all binding sites were trypsin sensitive, and ¹²⁵I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (K_D = 3 nM) that were trypsin sensitive, and ¹²⁵I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of ¹²⁵I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.