Structural and Functional Integration of the PLCγ Interaction Domains Critical for Regulatory Mechanisms and Signaling Deregulation

Abstract

SummaryMultidomain proteins incorporating interaction domains are central to regulation of cellular processes. The elucidation of structural organization and mechanistic insights into many of these proteins, however, remain challenging due to their inherent flexibility. Here, we describe the organization and function of four interaction domains in PLCγ1 using a combination of structural biology and biochemical approaches. Intramolecular interactions within the regulatory region center on the cSH2 domain, the only domain that also interacts with the PLC-core. In the context of fibroblast growth-factor receptor signaling, the coordinated involvement of nSH2 and cSH2 domains mediates efficient phosphorylation of PLCγ1 resulting in the interruption of an autoinhibitory interface by direct competition and, independently, dissociation of PLCγ1 from the receptor. Further structural insights into the autoinhibitory surfaces provide a framework to interpret gain-of-function mutations in PLCγ isoforms linked to immune disorders and illustrate a distinct mechanism for regulation of PLC activity by common interaction domains.

Dysfunction of phospholipase Cγ in immune disorders and cancer

“…ITC with mutated cSH2 and/or core polypeptides confirmed that the following residues are important at the cSH2/core interface: R748, R753, N728, S729 and D1019. Introducing substitutions in these residues into full length PLCγ and assaying for PLC activity in cells also showed that disrupting the cSH2/core interface imparted increased basal activity and increased EGF stimulated activity compared to wild type protein [29]. Therefore, a clearer picture of the molecular basis of auto-inhibition and activation emerges from these data ( Figure 1B).…”

“…Confirmation that the cSH2 is the domain in γSA directly involved in auto-inhibition came from NMR titration experiments [29]. The PLC-core from PLCγ was added independently into 15 N labelled domains of nSH2, cSH2 and spPH.…”

“…Nevertheless, other techniques are available that have provided insight into the structural arrangements of the γSA and its interactions with the core. For instance, a combination of nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS), crystallography (of fragments of the protein), isothermal titration calorimetry (ITC) and modelling were utilised to study PLCγ1 [29].…”

“…SAXS, a method that provides low-resolution structures of proteins in solution, was performed on the γSA, a number of tandem domains within the γSA and various tagged and complexed constructs [29]. Taking into account the SAXS and NMR data, a model was generated representing a possible average structural arrangement of the γSA, in which the spPH and cSH2 occupy the central lobe with nSH2 and SH3 peripheral to them ( Figure 1A, bottom panel).…”

“…However, the crystal structure of phosphorylated tandem nSH2-cSH2, showing that residues 781 to 790 bound in cis to the canonical cSH2 pY peptide binding pocket, was obtained only recently [29]. linker in the cSH2 binding site [29].…”