Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 ( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of 3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for 3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific 3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific 3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with gamma-32P]ATP and molybdate, the stimulated incorporation of the 32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that 3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.