Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells

Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4's antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes H-3-P4 binding to SIGCs and mimics P4's action, whereas exogenous hyaluronic acid binding protein attenuates P4's actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat, ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4's antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4's antiapoptotic action in granulosa cells.