Function Study Of The Truncated Ectodomain Of GPR48 In Osteoclast

GPR48, also known as LGR4, is a member of Rhodopsins family GPCRs, which is involved in regulation of many physiological functions. Recently it has been well recognized that loss of GPR48 results in developmental defects in many organs or tissues. Our previous study show that Gpr48-/- mice exhibit decrease of bone mass, and increase of osteoclast activation. Further study reveal that GPR48 negatively regulates osteoclastogeneis and bone resorption via competing with RANK for RANKL binding, which eventually blocks canonical RANKL-RANK pathway. In this study, we introduced the condition knockout mice model which can specifically knockout Gpr48 in osteoclast. Our results show that GPR48 deficiency in osteoclast will lead to hyper-activation of osteoclast and cause decrease of bone mass. RSPOs and Norrin are well known as the ligands for GPR48, we then wonder whether RSPOs or Norrin participate in osteoclastogenesis via GPR48. As results shown, it seems that GPR48 negatively regulation of osteoclast differentiation rarely rely on RSPOs or Norrin, while is related to RANKL/GPR48 stimulation of Gaq signal pathway.We also expressed and purified the ectodomain of GPR48 for treating Opg-/- mice, a kind of osteoporosis model, to figure out the possibility that whether GPR48 could be a potential drug for treating osteoclast related disease. Our results show that it can ameliorate the loss of bone mass. We’d further want to know the exact binding domain of GPR48 with RANKL. What’s more, we would like to find out the most efficiency fragments of GPR48 in inhibiting osteoclast differentiation. In this case, we utilized both the E.coli prokaryotic expression system and HEK293T mammalian cell expression system to express and purify the different truncated ectodomain proteins of GPR48. Our results show that deletion of LRRNT-LRR3 will not alter the ability of ectodomain of GPR48 in inhibiting RANKL induced osteoclast differentiation. Moreover, the efficiency of the binding ability between RANKL and GPR48 is much lower when ectodomain of GPR48 loss LRR14-LRR17 region.In conclusion, LRR3-LRR14 region of ectodomain of GPR48 perhaps play an important role in its interaction with RANKL and could be a potential drug for treating osteoclast related disease.