Specific Uptake of Retinoids into Human Promyelocytic Leukemia Cells HL‐60 by Retinoid‐specific Binding Protein: Possibly the True Retinoid Receptor

The uptake of all‐trans‐retinoic acid (RA) and two new retinoids [4‐(5,6,7,8‐tetrahydro‐5,5,8,8‐tetramethyl‐2‐naphthalenylcarbamoyl)benzoic acid (Am80) and (E)‐4‐[3‐(3,5‐di‐tert‐butylphenyl)‐3‐oxo‐1‐propenyl]benzoic acid (Ch55)] by HL‐60 human promyelocytic leukemia cells was investigated. For the investigation, [(3)H]RA and [(3)H]Am80 with high specific radioactivities (more than 50 Ci/mmol) were used. [(3)H]Am80 was prepared by hydrogenolysis of the corresponding chlorinated derivative of Am80 with tritium gas. The retinoids RA, Am80 and Ch55 were efficiently taken up by HL‐60 cells, and induced differentiation of the cells into mature granulocytes. The specific bindings (uptake) of RA, Am80 and Ch55 (the bindings inhibited competitively by the other two retinoids) by HL‐60 cells were due to a newly detected binding protein. The protein that bound specifically to RA appeared identical to that which bound specifically to Am80 by high‐performance liquid chromatography (HPLC), and was named retinoid‐specific binding protein (RSBP). One HL‐60 cell was found to contain about 1500 molecules of RSBP distributed between the nuclear fraction and cytosolic fraction in proportions of about 4:1. The bindings of the three retinoids (RA, Am80 and Ch55) to RSBP (i.e., formation of retinoid‐RSBP complexes) greatly enhanced the affinity of RSBP for the nuclei. The apparent molecular weight of RSBP was estimated to be 95,000 daltons by size exclusion HPLC. The association constants (Ka) of RSBP were calculated to be 2.4 × 10(10)M(‐1) for RA and 4.4 × 10(10)M(‐1) for Am80 from Scatchard plots. The bindings of RA, Am80 and Ch55 to RSBP were mutually competitive, indicating that the binding sites for RA, Am80 and Ch55 were identical. The very high affinities of these retinoids for RSBP (Ka's of the order of 10(10)M(‐1)) correspond to the effective concentrations of these retinoids in HL‐60 cell culture medium for induction of differentiation of the cells. The mutually competitive bindings of these retinoids strongly support the idea that RSBP is the true receptor of retinoids.