ISSN:
0730-2312
Keywords:
Scatchard analysis
;
dissociation kinetics
;
epidermal growth factor
;
binding analysis
;
Triton X-100 extract
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 × 104 sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 × 106 sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1. × 10-3s-1 (1.0-1.3 × 106 sites per cell) and a slow-dissociating site, with a k-1 of 3.5 × 10-5s-1 (0.6-0.7 × 106 sites per cell).The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that ∼5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 × 10-3s-1) and a slow-dissociating component (k-1 = 9.1 × 10-5s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4°C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4°C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240390411
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