Characterization of receptors for recombinant human tumor necrosis factor-α from human placental membranes

High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-α) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-α iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-α receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of ¹²⁵I-rhTNF-α to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37°C, 24°C and 4°C, respectively. However, the maximum binding obtainable at 4°C was only 40% of that at 37°C. The binding ¹²⁵I-rhTNF-α to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-α, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-β: previously called lymphotoxin). This is similar to the behavior of TNF-α receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-α and rhTNF-β bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,00 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-α are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-β.