WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex

Abstract

SummaryWe identified the WD-repeat-containing protein, WDR36, as an interacting partner of the b isoform of thromboxane A 2 receptor (TPb) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TPb by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TPb stimulation. TPb-WDR36 colocalization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but the complex translocated to intracellular vesicles following receptor stimulation. Coexpression of WDR36 and its siRNA-mediated knockdown, respectively, increased and inhibited TPb-induced Gaq signalling. Interestingly, WDR36 co-immunoprecipitated with Gaq, and promoted TPb-Gaq interaction. WDR36 also associated with phospholipase Cb (PLCb) and increased the interaction between Gaq and PLCb, but prevented sequestration of activated Gaq by GRK2. In addition, the presence of TPb in PLCb immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gaq-mediated signalling by TPb. We report that WDR36 acts as a new scaffold protein tethering a G-proteincoupled receptor, Gaq and PLCb in a signalling complex.

Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

“…β 2 AR and the yeast two-hybrid pAS2-1-TPβCT constructs were described previously (Parent et al. , 2009; Cartier et al. , 2011).…”

Structural Insights into Phospholipase C-βFunction

“…An intriguing possibility is that the distal CTD could also influence the membrane association of the catalytic core by inducing differences in local membrane curvature. An additional layer of regulatory complexity arises from the observation that PLCb isozymes interact with numerous scaffolding proteins to form signaling complexes (Cai et al, 2005;Cartier et al, 2011;Sun et al, 2013). How these higher order complexes contribute to PLCb regulation and whether they alter activation by Ga q , Gbg, and small GTPases are not understood, and represent the next frontier for structure/function analyses of PLCb enzymes.…”

“…Members of the Rho family of small molecular weight G proteins, such as the Rac isoforms, also directly bind and activate PLCb, linking PLCb activity to GPCR-independent signaling cascades (Gresset et al, 2012;Kadamur and Ross, 2013). It is also increasingly recognized that the membrane itself plays a role in the regulation of PLCb, as may interactions with scaffolding proteins (Cartier et al, 2011;Grubb et al, 2011Grubb et al, , 2012Sun et al, 2013). In this review, we highlight the current understanding of the molecular basis of regulation of mammalian PLCb enzymes and their modulation by small molecules, with an emphasis on recent structural discoveries.…”

STRAP Acts as a Scaffolding Protein in Controlling the TLR2/4 Signaling Pathway

“…Several WD-motif containing proteins, such as WDR36, WDR62, and Morg1 are involved in protein-protein interactions and participate in different biological functions12131415. In particular, STRAP plays a role in regulating the TGF-β signaling pathway, which is involved in cross-talk with the NF-κB signaling pathway616.…”