Structural and dynamic changes in P-Rex1 upon activation by PIP₃ and inhibition by IP₄

Area of Science:

• Biochemistry
• Cell Biology
• Structural Biology

Background:

• PIP3-dependent Rac exchanger 1 (P-Rex1) is a guanine-nucleotide exchange factor (GEF) crucial for neutrophil chemotaxis and cancer metastasis.
• P-Rex1 is activated by PIP3 and Gβγ subunits, but its precise regulation remains unclear.

Purpose of the Study:

• To elucidate the regulatory mechanisms of P-Rex1, particularly its inhibition by inositol phosphates.
• To determine the structural basis of P-Rex1 autoinhibition and activation.

Main Methods:

• Cryo-electron microscopy (Cryo-EM) to visualize the P-Rex1·IP4 complex.
• Biochemical assays to assess GEF activity.
• Mutagenesis studies to probe protein interfaces.
• Liposome binding assays to study membrane interactions.
• Analysis of P-Rex1 variants in chemokine-induced cell migration.

Main Results:

• Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 by inducing an autoinhibited conformation where the PH domain blocks the DH active site.
• This inhibited state is stabilized by interactions between DEP1-DH and PH-4HB domains.
• Disrupting these interfaces enhances P-Rex1 activity and alters its conformation.
• PIP3-containing liposomes disrupt these interfaces, increasing P-Rex1 dynamics and activity.

Conclusions:

• IP4 acts as an inhibitor of P-Rex1, contributing to the regulation of basal activity in neutrophils.
• The identified autoinhibited structure provides a mechanistic understanding of P-Rex1 regulation.
• P-Rex1 activity is dynamically regulated by the balance between inhibitory inositol phosphates and activating PIP3 signals.