In this study, we have provided biochemical and physiological evidence to demonstrate that widely expressed GRK5 and GRK6 have an unexpected and important function during Wnt signaling as membrane-bound kinases phosphorylating LRP6. The classical role of GRK5 and GRK6 is to phosphorylate activated GPCRs, initiating β-arrestin binding for receptor desensitization, internalization, and signaling (12
). Our findings that GRK5 and GRK6 phosphorylate the single membrane-spanning receptor LRP6 on defined serine/threonine sites (i.e.
serine 1490) within proline-rich PPPSP motifs and thereby activate LRP6 are important and interesting in two respects. First, this result extends the range of receptor families, in addition to GPCRs, that can be phosphorylated and regulated by GRK5 and GRK6. Furthermore, instead of having an inhibitory role as in classical G-protein mediated signaling, GRK5 and GRK6 might have a stimulatory role in Wnt/LRP6 signaling. This suggests that GRK5 and GRK6 may have much broader roles than are currently appreciated. Second, our data provide a novel mechanism for LRP6 phosphorylation and regulation at the plasma membrane. Previously, we have shown that β-arrestin2 is recruited to the plasma membrane to bind to Frizzled through Dishevelled and thereby regulates Frizzled function (17
). Because GRKs and β-arrestins function in concert in regulating GPCRs, it is quite surprising that GRK5/6 and β-arrestin2 participate in regulating Wnt signaling by separate means, delineating their distinct and independent functions. Additional studies will be needed to clarify how Wnt-induced activation of LRP6 leads to GRK5/6 recognition of the receptor as substrate.
In addition, the data presented here also solve a paradox within the current model of LRP6 phosphorylation by GSK3β in the cytoplasm. In a cellular system that included an artificially membrane-associated GSK3β mutant, cytosolic GSK3β was proposed to be recruited to the plasma membrane through the adaptor protein axin to phosphorylate LRP6 at the PPPSP motifs (11
). However, for axin to bind LRP6, the PPPSP motifs must first be phosphorylated, at a time when GSK3β is still in the cytoplasm (10
). Therefore, at least the initiation of both PPPSP phosphorylation and axin translocation to membrane LRP6 would be expected to be GSK3β independent. Our finding that GRK5 and GRK6 function as LRP6 kinases presents a straightforward mechanism for understanding LRP6 phosphorylation and activation, as these kinases are membrane bound already and are therefore available to initiate LRP6 by phosphorylation to promote subsequent axin/GSK3β binding. In addition, it has been recently shown that parathyroid hormone (PTH) and its receptor (PTHR), a GPCR that can be phosphorylated by GRK5 (31
), regulates phosphorylation of LRP6 serine 1490 to activate LRP6 in a Wnt-independent fashion (32
). However, the kinase responsible for PTH/PTHR-mediated LRP6 phosphorylation is unknown. Our studies might also suggest a role for GRK5 and GRK6 in PTH/PTHR-mediated LRP6 phosphorylation.
Taken together, our results provide new insight into the actions of GRK5 and GRK6. By phosphorylating LRP6 on PPPSP motifs they contribute to Wnt signaling, a pathway critical for basic biological processes in cells and during embryonic development.