The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Abstract

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G_q-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G_q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα_q to phospholipase C (PLC)-β and the thromboxane A₂ receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M₃ and bradykinin B₂ receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca²⁺]_i increases. Surprisingly, WDR36 also augmented M₃ receptor-induced [Ca²⁺]_i increases, which was due to increased Ca²⁺ mobilization while the Ca²⁺ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G_q-coupled M₃, B₂ and H₁ receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B₂ receptors. It is concluded that WDR36 reduces signalling of G_q-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα_q and PLCβ. Our results support a role of WDR36 in orchestration of G_q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.

Keywords: Ca(2+) signalling; G(q); G-protein-coupled receptors; Phospholipase C; Sphingosine kinase; WDR36.