*2.5. UDCA-LPE Serves as a Bivalent Ligand Bridging Between Integrin β1 and LPAR1*

We found that treatment of CL48 cells with UDCA-LPE or LPE was able to induce phosphorylation of b-Raf at Ser445 in the first 15 min (Figure 5A). It is known that LPE interacts with a G protein-coupled receptor LPAR1 [16] and that LPAR activation induces the activation of PKA [17,18]. We used anti-PKA substrates (RRXS\*/T\*) antibody to determine the activity of PKA. UDCA-LPE was able to rapidly induce phosphorylation of PKA substrates maximizing at 15 min (Figure 5B). The UDCA-LPE-induced activation of b-Raf (but not c-Raf) and ERK was inhibited by pre-treatment with PKA antagonist Rp-cAMP (Figure 5C). These data showed the ability of UDCA-LPE to trigger LPE/LPAR1 signalling via PKA/b-Raf/ERK pathways. We further performed immunoprecipitation of integrin β1 followed by immunoblotting with an anti-LPAR1 antibody. LPAR1 was nearly undetectable in the pull-downs of control cells whereas LPAR1 protein levels were markedly elevated in those of UDCA-LPE-treated cells (Figure 5D). Our results suggest that UDCA-LPE may act as a bivalent ligand bridging between integrins and LPAR1 to form a tri-component complex.

**Figure 5.** UDCA-LPE induces the LPE signalling and complex formation between LPAR1 and integrin β1. (**A**,**B**) CL48 cells were treated with (**A**,**B**) 90 μM UDCA-LPE or (**A**) 90 μM LPE for 1 min to 2 h. Lysates were probed with antibodies against (**A**) phospho-b-Raf (Ser445) and (**B**) PKA substrates (RRXS\*/T\*). (**C**) CL48 cells were treated with 200 μM Rp-cAMP for 30 min and 90 μM UDCA-LPE for 1 min to 15 min. Lysates were probed with antibodies against PKA substrate (RRXS \*/T \*), phospho-b-Raf (Ser445), phospho-c-Raf (Ser338) and phospho-ERK (Thr202/Tyr204). (**D**) CL48 cells were treated with 90 μM UDCA-LPE for 1 h. Integrin β1-containing proteins were immunoprecipitated with a polyclonal anti-integrin β1 antibody and immunoblotted using anti-LPAR1 or anti-integrin β1 antibody.
