Structural Insights into Formation of an Active Signaling Complex between Rac and Phospholipase C Gamma 2

Abstract

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.

Dysfunction of phospholipase Cγ in immune disorders and cancer

“…PLCγ mutations in immune disorders Table 2. PLCγ mutations in cancer been discovered that PLCγ2 is also activated by the small GTPase Rac [40,41]. by a phosphorylation independent mechanism (dotted arrow).…”

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling

“…These states were initially described for H-Ras as having low versus high affinity, respectively, toward Ras effectors (39). (ii) Stabilization of the Phe-897 side chain was in one of the two conformational states observed in the crystal structures of free spPH (25). Replacement of Phe-897 in human PLC␥ 2 spPH by the corresponding human PLC␥ 1 residue glutamine is expected to reduce the hydrophobic momentum of the Rac2 binding pocket on PLC␥ 2 spPH and, possibly, interfere with the reorientation of the Leu-67 side chain upon complex formation (Fig.…”

“…Mutational and structural analyses have shown that the stimulation of human PLC␥ 2 by activated Rac GTPases is due to an interaction of the Rac switch I and II regions with specific residues in the C-terminal half of the PLC␥ 2 spPH domain (24,25). Fig.…”

“…A residue unique for spPH of PLC␥ 2 , but not PLC␥ 1 , Phe-897, was found to be particularly important for the functional and structural PLC␥ 2 -spPH/Rac2 interaction. Replacement of Phe-897 to the corresponding glutamine residue of PLC␥ 1 , F897Q, did not affect the overall three-dimensional structure of the PLC␥ 2 spPH domain, but it specifically blocked the interaction of the mutant domain with activated Rac2 (24,25).…”

“…Unlike activation of PLC␤ 2 , which is mediated by Rac interacting with the N-terminal PH domain of this effector, activation of PLC␥ 2 involves binding of Rac to the bipartite, split PH domain (spPH) juxtaposed between the two halves, X and Y, of the PLC␥ 2 catalytic domain (24). The threedimensional structures of the heterodimeric complex between PLC␥ 2 spPH and GTP␥S-activated Rac2, monomeric spPH, and monomeric Rac2 liganded with either GTP␥S or GDP allowed us to elucidate the conformational changes that accompany the formation of the signaling active PLC␥ 2 -spPH/ Rac2 heterodimer (25). A residue unique for spPH of PLC␥ 2 , but not PLC␥ 1 , Phe-897, was found to be particularly important for the functional and structural PLC␥ 2 -spPH/Rac2 interaction.…”